Compositions and methods for enhanced mucosal delivery of parathyroid hormone

ABSTRACT

Pharmaceutical compositions and methods are described comprising at least a parathyroid hormone peptide (PTH) preferably PTH 1-34  and one or more mucosal delivery-enhancing agents for enhanced nasal mucosal delivery of PTH, for treating or preventing osteoporosis or osteopenia in a mammalian subject, preferably a human.

CROSS REFERENCE TO RELATED APPLICATIONS

This application is a continuation of prior U.S. application Ser. No.11/744,745, filed May 4, 2007, which is a continuation of prior U.S.application Ser. No. 11/126,996, filed May 10, 2005, which claimed thebenefit under 35 U.S.C. §119(e) of U.S. Provisional Application No.60/570,113, filed May 10, 2004, each of which is hereby incorporated byreference in its entirety.

SEQUENCE LISTING

This application includes a Sequence Listing submitted herewith viaEFS-Web as an ASCII file created on Mar. 16, 2010, namedMDR-04-04CON2_SeqList.txt, which is 1,882 bytes in size, and is herebyincorporated by reference in its entirety.

BACKGROUND OF THE INVENTION

The teachings of all the references cited in the present specificationare incorporated in their entirety by reference.

Osteoporosis can be defined as a systemic skeletal disease characterizedby low bone mass, microarchitectural deterioration of bone tissue, andincreased bone fragility and susceptibility to fracture. It mostcommonly affects older populations, primarily postmenopausal women.

The prevalence of osteoporosis poses a serious health problem. TheNational Osteoporosis Foundation has estimated that 44 million peopleare experiencing the effects of osteoporosis or osteopenia. By the year2010, osteoporosis will affect more than 52 million people and, by 2020,more than 61 million people. The prevalence of osteoporosis is greaterin Caucasians and Asians than in African-Americans, perhaps becauseAfrican-Americans have a higher peak bone mass. Women are affected ingreater numbers than men are because men have a higher peak bonedensity. Furthermore, as women age the rate of bone turnover increases,resulting in accelerated bone loss because of the lack of estrogen aftermenopause.

The goal of pharmacological treatment of osteoporosis is to maintain orincrease bone strength, to prevent fractures throughout the patient'slife, and to minimize osteoporosis-related morbidity and mortality bysafely reducing the risk of fracture. The medications that have beenused most commonly to treat osteoporosis include calcium, and vitamin D,estrogen (with or without progestin), bisphonates, selective estrogenreceptor modulators (SERMs), and calcitonin.

Parathyroid hormone (PTH) has recently emerged as a popular osteoporosistreatment. Unlike other therapies that reduce bone resorption, PTHincreases bone mass, which results in greater bone mineral density(BMD). PTH has multiple actions on bone, some direct and some indirect.PTH increases the rate of calcium release from bone into blood. Thechronic effects of PTH are to increase the number of bone cells bothosteoblasts and osteoclasts, and to increase the remodeling bone. Theseeffects are apparent within hours after PTH is administered and persistfor hours after PTH is withdrawn. PTH administered to osteoporoticpatients leads to a net stimulation of bone formation especially intrabecular bone in the spine and hip resulting in a highly significantreduction in fractures. The bone formation is believed to occur by thestimulation of osteoblasts by PTH as osteoblasts have PTH receptors.

Parathyroid hormone (PTH) is a secreted, 84 amino acid residuepolypeptide having the amino acid sequenceSer-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-PheVal Ala Leu Gly Ala Pro Leu Ala Pro Arg Asp Ala Gly Ser Gln Arg Pro ArgLys Lys Glu Asp Asn Val Leu Val Glu Ser His Glu Lys Ser Leu Gly Glu AlaAsp Lys Ala Asn Val Asp Val Leu Thr Lys Ala Lys Ser Gln (SEQ ID NO: 1).Studies in humans with certain forms of PTH have demonstrated ananabolic effect on bone, and have prompted significant interest in itsuse for the treatment of osteoporosis and related bone disorders.

Using the N-terminal 34 amino acids of the bovine and human hormoneSer-Val-Ser-Glu-Ile-Gln-Leu-Met-His-Asn-Leu-Gly-Lys-His-Leu-Asn-Ser-Met-Glu-Arg-Val-Glu-Trp-Leu-Arg-Lys-Lys-Leu-Gln-Asp-Val-His-Asn-Phe(SEQ ID NO: 2) for example, which by all published accounts are deemedbiologically equivalent to the full length hormone, it has beendemonstrated in humans that parathyroid hormone enhances bone growthparticularly when administered in pulsatile fashion by the subcutaneousroute. A slightly different form of PTH, human PTH(1-38) has shownsimilar results.

PTH preparations have been reconstituted from fresh or lyophilizedhormone, and incorporate various forms of carrier, excipient andvehicle. Most are prepared in water-based vehicles such as saline, orwater acidified typically with acetic acid to solubilize the hormone.The majority of reported formulations also incorporate albumin as astabilizer. See for example, Reeve et al., Br. Med. J. 280:6228, 1980;Reeve et al., Lancet 1:1035, 1976; Reeve et al., Calcif. Tissue Res.21:469, 1976; Hodsman et al., Bone Miner 9(2):137, 1990; Tsai et al., J.Clin. Endocrinol Metab. 69(5):1024, 1989; Isaac et al., Horm. Metab.Res. 12(9):487, 1980; Law et al., J. Clin Invest. 72(3):1106, 1983; andHulter, J. Clin Hypertens 2(4):360, 1986. Other reported formulationshave incorporated an excipient such as mannitol, which is present eitherwith the lyophilized hormone or in the reconstitution vehicle.

PTH1-34 also called teriparatide is currently on the market under thebrand name FORTEO®, Eli Lilly, Indianapolis, Ind. for the treatment ofpostmenopausal women with osteoporosis who are at high risk of fracture.This drug is administered by a once daily subcutaneous injection of 20μg in a solution containing acetate buffer, mannitol, and m-cresol inwater, pH 4. However, many people are adverse to injections, and thusbecome non-compliant with the prescribed dosing of the PTH. Thus, thereis a need to develop an intranasal formulation of a parathyroid hormonepeptide that has suitable bioavailability such that therapeutic levelscan be achieved in the blood to be effective to treat osteoporosis orosteopenia. FORTEO® is manufactured by recombinant DNA technology usingan Escherichia coli strain. PTH₁₋₃₄ has a molecular weight of 4117.87daltons. Reviews on PTH₁₋₃₄ and its clinical that have been published,including, e.g., Brixen et al., 2004; Dobnig, 2004; Eriksen and Robins,2004; Quattrocchi and Kourlas 2004, are hereby incorporated byreference. Forsteo is currently licensed in the United States (asFORTEO®) and Europe. The safety of teriparatide has been evaluated inover 2800 patients in doses ranging from 5 to 100 μg per day in shortterm trials. Doses of up to 40 μg per day have been given for up to twoyears in long term trials. Adverse events associated with Forsteo wereusually mild and generally did not require discontinuation of therapy.The most commonly reported adverse effects were dizziness, leg cramps,nausea, vomiting and headache. Mild transient hypercalcemia has beenreported with Forsteo which is usually self limiting within 6 hours.

Teriparatide has been previously been administered intranasally tohumans at doses of up to 500 μg per day for 7 days in one study (SuntoryNews Release, Suntory Establishes Large Scale Production of recombinanthuman PTH₁₋₃₄ and obtains promising results from Phase 1 Clinical Trialsusing a Nasal Formulation. February 1999,http://www.suntory.com/news/1999-02.html accessed 15 Apr. 2004) and inanother study subjects received up to 1,000 μg per day for 3 months(Matsumoto et al., “Daily Nasal Spray of hPTH₁₋₃₄ for 3 Months IncreasesBone Mass In Osteoporotic Subjects,” (ASBMR 2004 presentation 1171 Oct.4, 2004, Seattle Wash.). No safety concerns were noted with this route.

Currently Forsteo is administered as a daily subcutaneous injection. Itwould be preferable for patient acceptability if a non-injected route ofadministration were available, including nasal, buccal, gastrointestinaland dermal.

DISCLOSURE OF THE INVENTION

Preferably the parathyroid hormone and the mammal is a human. In a mostpreferred embodiment the parathyroid hormone peptide, is PTH₁₋₃₄, alsoknown as teriparatide. Tregear, U.S. Pat. No. 4,086,196, described humanPTH analogues and claimed that the first 27 to 34 amino acids are themost effective in terms of the stimulation of adenylyl cyclase in an invitro cell assay. Pang et al., WO 93/06845, published Apr. 15, 1993,described analogues of hPTH which involve substitutions of Arg²⁵, Lys²⁶,Lys²⁷ with numerous amino acids, including alanine, asparagine, asparticacid, cysteine, glutamine, glutamic acid, glycine, histidine,isoleucine, leucine, methionine, phenylalanine, proline, serine,threonine, tryptophan, tyrosine, or valine. Other PTH analogues aredisclosed in the following patents, hereby incorporated by reference:U.S. Pat. No. 5,317,010; U.S. Pat. No. 4,822,609; U.S. Pat. No.5,693.616; U.S. Pat. No. 5,589,452; U.S. Pat. No. 4,833,125; U.S. Pat.No. 5,607,915; U.S. Pat. No. 5,556,940; U.S. Pat. No. 5,382,658; U.S.Pat. No. 5,407,911; U.S. Pat. No. 6,583,114; U.S. Pat. No. 6,541,450;U.S. Pat. No. 6,376,502; U.S. Pat. No. 5,955,425; U.S. Pat. No.6,316,410; U.S. Pat. No. 6,110,892; U.S. Pat. No. 6,051,686; U.S. Pat.No. 5,695,955; U.S. Pat. No. 4,771,124; and U.S. Pat. No. 6,376,502.

PTH operates through activation of two second messenger systems,G_(s)-protein activated adenylyl cyclase (AC) and G_(q)-proteinactivated phospholipase C_(β). The latter results in a stimulation ofmembrane-bound protein kinase Cs (PKC) activity. The PKC activity hasbeen shown to require PTH residues 29 to 32 (Jouishomme et al., J. BoneMineral Res. 9:1179-1189, 1994. It has been established that theincrease in bone growth, i.e. that effect which is useful in thetreatment of osteoporosis, is coupled to the ability of the peptidesequence to increase AC activity. The native PTH sequence has been shownto have all of these activities. The hPTH-(1-34) sequence is typicallyshown as:

(SEQ ID NO: 2) Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys HisLeu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val HisAsn Phe.

The following linear analogue, hPTH₁₋₃₁NH₂, has only AC-stimulatingactivity and has been shown to be fully active in the restoration ofbone loss in the ovariectomized rat model [Rixon, R. H. et al., J. BoneMiner. Res. 9:1179-1189, 1994; Whitfield et al., Calcified Tissue Int.58:81-87, 1996]; Willick et al., U.S. Pat. No. 5,556,940, herebyincorporated by reference:

(SEQ ID NO: 3) Ser Val Ser Glu Ile Gln Leu Met His Asn Leu Gly Lys HisLeu Asn Ser Met Glu Arg Val Glu Trp Leu Arg Lys Lys Leu Gln Asp Val.

The above molecule, SEQ ID NO: 3, and its counterpart with a Leu₂₇substitution SEQ ID NO:2 may have a free carboxyl ending instead of theamide ending. Another PTH analog is [Leu₂₇]cyclo(Glu₂₂-Lys₂₆)PTH₁₋₃₁.

In other embodiments of the present invention, the PTH composition isadministered in droplets exiting from an actuator form a spray plumewith a measured ellipsoid (ratio of length of longest to shortest axes)of 1-2,the droplets exiting from the actuator form a spray plume with ameasured ellipsoid (ratio of length of longest to shortest axes) of1-1.3, the volume median droplet size is between 10 and 1000 microns(10<Dv,50<1000), where the Dv,50 is between 30 and 300 microns, thepercentage of droplets having a diameter 10 microns or less is 10% orless and the percentage of droplets having a diameter 10 microns or lessis 1% or less.

The present invention is also directed to an intranasal formulation of aPTH-agonist that is substantially free of proteins or polypeptides thatstabilize the formulation. In particular, the preferred formulation isfree of such proteins as albumin, and collagen-derived proteins such asgelatin.

In other aspects of the present invention a transmucosal PTH peptideformulation is comprised of a PTH peptide, water and a solubilizingagent having a pH of 3-6.5. In a preferred embodiment, thesolubilization agent is a cyclodextrin.

In another embodiment of the present invention a transmucosal PTHpeptide formulation is comprised of a PTH peptide, water, a solubilizingagent, preferably a cyclodextrin, and at least one polyol, preferably 2polyols. In alternate embodiments the formulation may contain one or allof the following: a chelating agent, a surface-acting agent and abuffering agent.

In another embodiment of the present invention the formulation iscomprised of a PTH peptide, water, chelating agent and a solubilizationagent.

In another embodiment of the present invention the formulation iscomprised of a PTH peptide, water and a chelating agent having a pH of3-6.5.

In another embodiment of the present invention the formulation iscomprised of a PTH peptide, water, chelating agent and at least onepolyol, preferably two polyols. Additional embodiments may include oneor more of the following: a surface-active agent, a solubilizing agentand a buffering agent.

In another embodiment of the present invention the formulation iscomprised of a PTH peptide, water, and at least two polyols, such aslactose and sorbitol. Additional agents, which can be added to theformulation, include, but are not limited to, a solubilization agent, achelating agent, one or more buffering agents and a surface-actingagent.

The enhancement of intranasal delivery of a PTH peptide agonistaccording to the methods and compositions of the invention allows forthe effective pharmaceutical use of these agents to treat a variety ofdiseases and conditions in mammalian subjects.

The present invention fills this need by providing for a liquid ordehydrated PTH peptide formulation wherein the formulation issubstantially free of a stabilizer that is a polypeptide or a protein.The liquid parathyroid hormone formulation is comprised of water,parathyroid hormone and at least one of the following additives selectedfrom the group consisting of polyols, surface-active agents,solubilizing agents and chelating agents. The pH of the formulation ispreferably 3 to about 7.0, preferably 4.5 to about 6.0, most preferablyabout 5.0±0.3.

Another embodiment of the present invention is an aqueous PTHformulation of the present invention is comprised of water, a PTHpeptide, a polyol and a surface-active agent wherein the formulation hasa pH of about 3 to about 6.5, and the formulation is substantially freeof a stabilizer that is a protein or polypeptide.

Another embodiment of the present invention is an aqueous PTH peptideformulation comprised of water, PTH peptide, a polyol and a solubilizingagent wherein the formulation has a pH of about 3.0 to about 6.5, andthe formulation is substantially free of a stabilizer that is a proteinor polypeptide.

Another embodiment of the present invention is an aqueous PTH peptideformulation comprised of water, PTH peptide, a solubilizing agent and asurface-active agent wherein the formulation has a pH of about 3.0 toabout 6.5, and the formulation is substantially free of a stabilizerthat is a protein or polypeptide.

Another embodiment of the invention is an aqueous PTH peptideformulation comprised of water, a PTH peptide, a solubilizing agent, apolyol and a surface-active agent wherein the formulation has a pH ofabout 3.0 to about 6.5, and the formulation is substantially free of astabilizer that is a protein or polypeptide.

In another aspect of the present invention, the stable aqueousformulation is dehydrated to produce a dehydrated PTH peptideformulation comprised of PTH peptide and at least one of the followingadditives selected from the group consisting of polyols, surface-activeagents, solubilizing agents and chelating agents, wherein saiddehydrated PTH peptide formulation is substantially free of a stabilizerthat is a protein or polypeptide such as albumin, collagen orcollagen-derived protein such as gelatin. The dehydration can beachieved by various means such as lyophilization, spray-drying,salt-induced precipitation and drying, vacuum drying, rotaryevaporation, or supercritical CO₂ precipitation.

In one embodiment, the dehydrated PTH peptide is comprised of PTHpeptide, a polyol and a solubilizing agent, wherein the formulation issubstantially free of a stabilizer that is a protein.

In another embodiment, the dehydrated PTH peptide formulation iscomprised of a PTH peptide, a polyol, and a surface-active agent whereinthe PTH peptide formulation is substantially free of a stabilizer thatis a protein or polypeptide.

In another embodiment, the dehydrated PTH peptide formulation iscomprised of a PTH peptide, a surface-active agent, and a solubilizingagent wherein the PTH peptide formulation is substantially free of astabilizer that is a protein or polypeptide.

In another embodiment of the present invention, the dehydrated PTHpeptide formulation is comprised of a PTH peptide, a polyol, asurface-active agent and a solubilizing agent wherein the PTH peptideformulation is substantially free of a stabilizer that is a protein orpolypeptide.

Another aspect of the present invention is an intranasal PTH peptideformulation contain within an actuator able to produce an aerosol ofsaid solution, wherein the spray pattern ellipticity ratio of saidaerosol is between 1.00 and 1.40 when measured at a height of between0.5 cm and 10 cm distance from the actuator tip, which has preferably anellipticity of between 1.00 and 1.30 and produces an aerosol of between20 and 200 microliters per actuation.

In another embodiment, the intranasal PTH peptide solution is in anactuator, which produces an aerosol of said solution, wherein the spraypattern major and minor axes of said aerosol are between 10 and 50 mmwhen measured at a height of between 0.5 cm and 10 cm distance from theactuator tip. In another embodiment, an aqueous solution of a PTHpeptide is in a container attached an actuator so that an aerosol of thesolution is produced wherein less than 10% of the droplets produced aresmaller than 10 microns in size and aerosol containing the PTH peptidecontains 20 and 200 microliters solution per actuation. In anotherembodiment, a solution of the PTH peptide is in a container attached toan actuator so that the aerosol of the solution produced upon actuationhas droplets between 25 and 700 microns.

Any solubilizing agent can be used but a preferred one is selected fromthe group consisting of hydroxypropyl-β-cyclodextran,sulfobutylether-β-cyclodextran, methyl-β-cyclodextrin and chitosan.

Generally a polyol is selected from the group consisting of lactose,sorbitol, trehalose, sucrose, mannose and maltose and derivatives andhomologs thereof.

A satisfactory surface-active agent is selected from the groupconsisting of L-α-phosphatidylcholine didecanoyl (DDPC), polysorbate 20(Tween 20), polysorbate 80 (Tween 80), polyethylene glycol (PEG), cetylalcohol, polyvinylpyrolidone (PVP), polyvinyl alcohol (PVA), lanolinalcohol, and sorbitan monooleate.

In a preferred formulation, the PTH peptide formulation is alsocomprised of a chelating agent such as ethylene diamine tetraacetic acid(EDTA) or ethylene glycol tetraacetic acid (EGTA). Also a preservativesuch as chlorobutanol, methyl paraben, propyl paraben, butyl paraben,benzalkonium chloride, benzethonium chloride, sodium benzoate, sorbicacid, phenol, or ortho-, meta- or paracresol.

The pH is generally regulated using a buffer such as sodium citrate andcitric acid, and sodium acetate and acetic acid. An alternative bufferwould be acetic acid and sodium acetate or succinic acid and sodiumhydroxide.

The present invention also comprehends a formulation wherein theconcentration of the PTH peptide is 0.1-15.0 mg/mL, preferably 1.0-2mg/mL and the pH of the aqueous solution is 3.0-6.5 preferably about5.0±0.3.

The present invention further includes PTH peptide formulation whereinthe concentration of the polyol is between about 0.1% and 10% (w/v) andadditionally wherein the concentration of the polyol is in the rangefrom about 0.1% to about 3% (w/v).

The instant invention also includes a formulation, wherein theconcentration of the surface-active agent is between about 0.00001% andabout 5% (w/v), and wherein the concentration of the surface-activeagent is between about 0.0002% and about 0.1% (w/v).

The instant invention also includes a formulation, wherein theconcentration of the solubilzation agent is 1% -10% (w/v), and whereinthe concentration of the solubilizing agent is 2% to 5% (w/v).

The finished solution can be filtered and freeze-dried, lyophilized,using methods well known to one of ordinary skill in the art, and byfollowing the instructions of the manufacturer of the lyophilizingequipment. This produces a dehydrated PTH peptide formulationsubstantially free of a stabilizer that is a protein.

In another embodiment of the present invention, a PTH peptideformulation is comprised of an PTH peptide and a pharmaceuticallyacceptable carrier wherein the PTH-bind peptide formulation has at least1%, preferably 3% and most preferably at least 6% higher permeation inan in vitro tissue permeation assay than a control formulationconsisting of water, sodium chloride, a buffer and the PTH peptide, asdetermined by the transepithelial electrical resistance assay shown inExamples 2 and 7. In a preferred embodiment, the PTH formulation isfurther comprised of at least one excipient selected from the groupconsisting of a surface-active agent, a solubilization agent, a polyol,and a chelating agent.

In exemplary embodiments, the enhanced delivery methods and compositionsof the present invention provide for therapeutically effective mucosaldelivery of the PTH peptide agonist for prevention or treatment ofosteoporosis or osteopenia in mammalian subjects. In one aspect of theinvention, pharmaceutical formulations suitable for intranasaladministration are provided that comprise a therapeutically effectiveamount of a PTH peptide and one or more intranasal delivery-enhancingagents as described herein, which formulations are effective in a nasalmucosal delivery method of the invention to prevent the onset orprogression of osteoporosis or osteopenia in a mammalian subject. Nasalmucosal delivery of a therapeutically effective amount of a PTH peptideagonist and one or more intranasal delivery-enhancing agents yieldselevated therapeutic levels of the PTH peptide agonist in the subjectand promotes the increase in bone mass in an individual.

The enhanced delivery methods and compositions of the present inventionprovide for therapeutically effective mucosal delivery of a PTH peptidefor prevention or treatment of osteoporosis or osteopenia in mammaliansubjects. PTH peptide can be administered via a variety of mucosalroutes, for example by contacting the PTH peptide to a nasal mucosalepithelium, a bronchial or pulmonary mucosal epithelium, the oral buccalsurface or the oral and small intestinal mucosal surface. In exemplaryembodiments, the methods and compositions are directed to or formulatedfor intranasal delivery (e.g., nasal mucosal delivery or intranasalmucosal delivery).

In one aspect of the invention, pharmaceutical formulations suitable forintranasal administration are provided that comprise a therapeuticallyeffective amount of a PTH peptide agonist and one or more intranasaldelivery-enhancing agents as described herein, which formulations areeffective in a nasal mucosal delivery method of the invention to preventor treat osteoporosis.

In another aspect of the invention, pharmaceutical formulations andmethods are directed to administration of a PTH peptide agonist incombination with calcium, vitamin D, bisphosphonates, calcitonin or abone morphogenic protein. See U.S. Pat. No. 5,616,560 and U.S. Pat. No.5,700,774, hereby incorporated by reference.

The foregoing mucosal PTH peptide formulations and preparative anddelivery methods of the invention provide improved mucosal delivery of aPTH peptide to mammalian subjects. These compositions and methods caninvolve combinatorial formulation or coordinate administration of one ormore PTH peptides with one or more mucosal delivery-enhancing agents.Among the mucosal delivery-enhancing agents to be selected from toachieve these formulations and methods are (A) solubilization agents;(B) charge modifying agents; (C) pH control agents; (D) degradativeenzyme inhibitors; (E) mucolytic or mucus clearing agents; (F)ciliostatic agents; (G) membrane penetration-enhancing agents (e.g., (i)a surfactant, (ii) a bile salt, (iii) a phospholipid or fatty acidadditive, mixed micelle, liposome, or carrier, (iv) an alcohol, (v) anenamine, (iv) an NO donor compound, (vii) a long-chain amphipathicmolecule (viii) a small hydrophobic penetration enhancer; (ix) sodium ora salicylic acid derivative; (x) a glycerol ester of acetoacetic acid(xi) a cyclodextrin or beta-cyclodextrin derivative, (xii) amedium-chain fatty acid, (xiii) a chelating agent, (xiv) an amino acidor salt thereof, (xv) an N-acetylamino acid or salt thereof, (xvi) anenzyme degradative to a selected membrane component, (xvii) an inhibitorof fatty acid synthesis, (xviii) an inhibitor of cholesterol synthesis;or (xiv) any combination of the membrane penetration enhancing agents of(i)-(xviii)); (H) modulatory agents of epithelial junction physiology,such as nitric oxide (NO) stimulators, chitosan, and chitosanderivatives; (I) vasodilator agents; (J) selective transport-enhancingagents; (K) stabilizing delivery vehicles, carriers, supports orcomplex-forming species with which the PTH peptide (s) is/areeffectively combined, associated, contained, encapsulated or bound tostabilize the active agent for enhanced mucosal delivery; and (L)alcohols such as ethanol.

In various embodiments of the invention, a PTH peptide is combined withone, two, three, four or more of the mucosal delivery-enhancing agentsrecited in (A)-(K), above.

These mucosal delivery-enhancing agents may be admixed, alone ortogether, with the PTH peptide, or otherwise combined therewith in apharmaceutically acceptable formulation or delivery vehicle. Formulationof a PTH peptide with one or more of the mucosal delivery-enhancingagents according to the teachings herein (optionally including anycombination of two or more mucosal delivery-enhancing agents selectedfrom (A)-(K) above) provides for increased bioavailability of the PTHpeptide following delivery thereof to a mucosal surface of a mammaliansubject.

Thus, the present invention is a method for treating osteoporosis orosteopenia in a mammal comprising transmucosally administering aformulation comprised of a PTH peptide, such that when at 50 μg of thePTH is administered transmucosally to the mammal the concentration ofthe PTH peptide in the plasma of the mammal increases by at least 5pmol, preferably at least 10 pmol per liter of plasma.

Intranasal delivery-enhancing agents are employed which enhance deliveryof PTH into or across a nasal mucosal surface. For passively absorbeddrugs, the relative contribution of paracellular and transcellularpathways to drug transport depends upon the pKa, partition coefficient,molecular radius and charge of the drug, the pH of the luminalenvironment in which the drug is delivered, and the area of theabsorbing surface. The intranasal delivery-enhancing agent of thepresent invention may be a pH control agent. The pH of thepharmaceutical formulation of the present invention is a factoraffecting absorption of PTH via paracellular and transcellular pathwaysto drug transport. In one embodiment, the pharmaceutical formulation ofthe present invention is pH adjusted to between about pH 3.0 to 6.5. Ina further embodiment, the pharmaceutical formulation of the presentinvention is pH adjusted to between about pH 3.0 to 5.0. In a furtherembodiment, the pharmaceutical formulation of the present invention ispH adjusted to between about pH 4.0 to 5.0. Generally, the pH is5.0±0.3.

As noted above, the present invention provides improved methods andcompositions for mucosal delivery of PTH peptide to mammalian subjectsfor treatment or prevention of osteoporosis or osteopenia. Examples ofappropriate mammalian subjects for treatment and prophylaxis accordingto the methods of the invention include, but are not restricted to,humans and non-human primates, livestock species, such as horses,cattle, sheep, and goats, and research and domestic species, includingdogs, cats, mice, rats, guinea pigs, and rabbits.

In order to provide better understanding of the present invention, thefollowing definitions are provided:

According to the present invention a parathyroid hormone peptide alsoincludes the free bases, acid addition salts or metal salts, such aspotassium or sodium salts of the peptides, and parathyroid hormonepeptides that have been modified by such processes as amidation,glycosylation, acylation, sulfation, phosphorylation, acetylation,cyclization and other well known covalent modification methods.

Osteopenia is a decreased calcification or density of bone, adescriptive term applicable to all skeletal systems in which thecondition is noted.

“Mucosal delivery enhancing agents” are defined as chemicals and otherexcipients that, when added to a formulation comprising water, saltsand/or common buffers and PTH peptide (the control formulation) producea formulation that produces a significant increase in transport of PTHpeptide across a mucosa as measured by the maximum blood, serum, orcerebral spinal fluid concentration (C_(max)) or by the area under thecurve, AUC, in a plot of concentration versus time. A mucosa includesthe nasal, oral, intestional, buccal, bronchopulmonary, vaginal, andrectal mucosal surfaces and in fact includes all mucus-secretingmembranes lining all body cavities or passages that communicate with theexterior. Mucosal delivery enhancing agents are sometimes calledcarriers.

“Non-infused administration” means any method of delivery that does notinvolve an injection directly into an artery or vein, a method whichforces or drives (typically a fluid) into something and especially tointroduce into a body part by means of a needle, syringe or otherinvasive method. Non-infused administration includes subcutaneousinjection, intramuscular injection, intraparitoneal injection and thenon-injection methods of delivery to a mucosa.

As noted above, the instant invention provides improved and usefulmethods and compositions for nasal mucosal delivery of a PTH peptide toprevent and treat osteoporosis or osteopenia in mammalian subjects. Asused herein, prevention and treatment of osteoporosis or osteopeniameans prevention of the onset or lowering the incidence or severity ofclinical osteoporosis by reducing increasing bone mass, decreasing boneresporption or reducing the incidence of fractured bones in a patient.

The PTH peptide can also be administered in conjunction with othertherapeutic agents such as bisphonates, calcium, vitamin D, estrogen orestrogen-receptor binding compounds, selective estrogen receptormodulators (SERMs), bone morphogenic proteins or calcitonin.

Improved methods and compositions for mucosal administration of PTHpeptide to mammalian subjects optimize PTH peptide dosing schedules. Thepresent invention provides mucosal delivery of PTH peptide formulatedwith one or more mucosal delivery-enhancing agents wherein PTH peptidedosage release is substantially normalized and/or sustained for aneffective delivery period of PTH peptide release ranges fromapproximately 0.1 to 2.0 hours; 0.4 to 1.5 hours; 0.7 to 1.5 hours; or0.8 to 1.0 hours; following mucosal administration. The sustainedrelease of PTH peptide achieved may be facilitated by repeatedadministration of exogenous PTH peptide utilizing methods andcompositions of the present invention.

Improved compositions and methods for mucosal administration of PTHpeptide to mammalian subjects optimize PTH peptide dosing schedules. Thepresent invention provides improved mucosal (e.g., nasal) delivery of aformulation comprising PTH peptide in combination with one or moremucosal delivery-enhancing agents and an optional sustainedrelease-enhancing agent or agents. Mucosal delivery-enhancing agents ofthe present invention yield an effective increase in delivery, e.g., anincrease in the maximal plasma concentration (C_(max)) to enhance thetherapeutic activity of mucosally-administered PTH peptide. A secondfactor affecting therapeutic activity of PTH peptide in the blood plasmaand CNS is residence time (RT). Sustained release-enhancing agents, incombination with intranasal delivery-enhancing agents, increase C_(max)and increase residence time (RT) of PTH peptide. Polymeric deliveryvehicles and other agents and methods of the present invention thatyield sustained release-enhancing formulations, for example,polyethylene glycol (PEG), are disclosed herein. The present inventionprovides an improved PTH peptide delivery method and dosage form fortreatment or prevention of osteoporosis or osteopenia in mammaliansubjects.

Within the mucosal delivery formulations and methods of the invention,the PTH peptide is frequently combined or coordinately administered witha suitable carrier or vehicle for mucosal delivery. As used herein, theterm “carrier” means a pharmaceutically acceptable solid or liquidfiller, diluent or encapsulating material. A water-containing liquidcarrier can contain pharmaceutically acceptable additives such asacidifying agents, alkalizing agents, antimicrobial preservatives,antioxidants, buffering agents, chelating agents, complexing agents,solubilizing agents, humectants, solvents, suspending and/orviscosity-increasing agents, tonicity agents, wetting agents or otherbiocompatible materials. A tabulation of ingredients listed by the abovecategories, can be found in the U.S. Pharmacopeia National Formulary,1990, pp 1857-1859. Some examples of the materials which can serve aspharmaceutically acceptable carriers are sugars, such as lactose,glucose and sucrose; starches such as corn starch and potato starch;cellulose and its derivatives such as sodium carboxymethyl cellulose,ethyl cellulose and cellulose acetate; powdered tragacanth; malt;gelatin; talc; excipients such as cocoa butter and suppository waxes;oils such as peanut oil, cottonseed oil, safflower oil, sesame oil,olive oil, corn oil and soybean oil; glycols, such as propylene glycol;polyols such as glycerin, sorbitol, mannitol and polyethylene glycol;esters such as ethyl oleate and ethyl laurate; agar; buffering agentssuch as magnesium hydroxide and aluminum hydroxide; alginic acid;pyrogen free water; isotonic saline; Ringer's solution, ethyl alcoholand phosphate buffer solutions, as well as other non toxic compatiblesubstances used in pharmaceutical formulations. Wetting agents,emulsifiers and lubricants such as sodium lauryl sulfate and magnesiumstearate, as well as coloring agents, release agents, coating agents,sweetening, flavoring and perfuming agents, preservatives andantioxidants can also be present in the compositions, according to thedesires of the formulator. Examples of pharmaceutically acceptableantioxidants include water soluble antioxidants such as ascorbic acid,cysteine hydrochloride, sodium bisulfite, sodium metabisulfite, sodiumsulfite and the like; oil-soluble antioxidants such as ascorbylpalmitate, butylated hydroxyanisole (BHA), butylated hydroxytoluene(BHT), lecithin, propyl gallate, alpha-tocopherol and the like; andmetal-chelating agents such as citric acid, ethylenediamine tetraaceticacid (EDTA), sorbitol, tartaric acid, phosphoric acid and the like. Theamount of active ingredient that can be combined with the carriermaterials to produce a single dosage form will vary depending upon theparticular mode of administration.

Within the mucosal delivery compositions and methods of the invention,various delivery-enhancing agents are employed which enhance delivery ofPTH peptide into or across a mucosal surface. In this regard, deliveryof PTH peptide across the mucosal epithelium can occur “transcellularly”or “paracellularly.” The extent to which these pathways contribute tothe overall flux and bioavailability of the PTH peptide depends upon theenvironment of the mucosa, the physico-chemical properties the activeagent, and on the properties of the mucosal epithelium. Paracellulartransport involves only passive diffusion, whereas transcellulartransport can occur by passive, facilitated or active processes.Generally, hydrophilic, passively transported, polar solutes diffusethrough the paracellular route, while more lipophilic solutes use thetranscellular route. Absorption and bioavailability (e.g., as reflectedby a permeability coefficient or physiological assay), for diverse,passively and actively absorbed solutes, can be readily evaluated, interms of both paracellular and transcellular delivery components, forany selected PTH peptide within the invention. For passively absorbeddrugs, the relative contribution of paracellular and transcellularpathways to drug transport depends upon the pKa, partition coefficient,molecular radius and charge of the drug, the pH of the luminalenvironment in which the drug is delivered, and the area of theabsorbing surface. The paracellular route represents a relatively smallfraction of accessible surface area of the nasal mucosal epithelium. Ingeneral terms, it has been reported that cell membranes occupy a mucosalsurface area that is a thousand times greater than the area occupied bythe paracellular spaces. Thus, the smaller accessible area, and thesize- and charge-based discrimination against macromolecular permeationwould suggest that the paracellular route would be a generally lessfavorable route than transcellular delivery for drug transport.Surprisingly, the methods and compositions of the invention provide forsignificantly enhanced transport of biotherapeutics into and acrossmucosal epithelia via the paracellular route. Therefore, the methods andcompositions of the invention successfully target both paracellular andtranscellular routes, alternatively or within a single method orcomposition.

As used herein, “mucosal delivery-enhancing agents” include agents whichenhance the release or solubility (e.g., from a formulation deliveryvehicle), diffusion rate, penetration capacity and timing, uptake,residence time, stability, effective half-life, peak or sustainedconcentration levels, clearance and other desired mucosal deliverycharacteristics (e.g., as measured at the site of delivery, or at aselected target site of activity such as the bloodstream or centralnervous system) of PTH peptide or other biologically active compound(s).Enhancement of mucosal delivery can thus occur by any of a variety ofmechanisms, for example by increasing the diffusion, transport,persistence or stability of PTH peptide, increasing membrane fluidity,modulating the availability or action of calcium and other ions thatregulate intracellular or paracellular permeation, solubilizing mucosalmembrane components (e.g., lipids), changing non-protein and proteinsulfhydryl levels in mucosal tissues, increasing water flux across themucosal surface, modulating epithelial junctional physiology, reducingthe viscosity of mucus overlying the mucosal epithelium, reducingmucociliary clearance rates, and other mechanisms.

As used herein, a “mucosally effective amount of PTH peptide”contemplates effective mucosal delivery of PTH peptide to a target sitefor drug activity in the subject that may involve a variety of deliveryor transfer routes. For example, a given active agent may find its waythrough clearances between cells of the mucosa and reach an adjacentvascular wall, while by another route the agent may, either passively oractively, be taken up into mucosal cells to act within the cells or bedischarged or transported out of the cells to reach a secondary targetsite, such as the systemic circulation. The methods and compositions ofthe invention may promote the translocation of active agents along oneor more such alternate routes, or may act directly on the mucosal tissueor proximal vascular tissue to promote absorption or penetration of theactive agent(s). The promotion of absorption or penetration in thiscontext is not limited to these mechanisms.

As used herein “peak concentration (C_(max)) of PTH peptide in a bloodplasma”, “area under concentration vs. time curve (AUC) of PTH peptidein a blood plasma”, “time to maximal plasma concentration (t_(max)) ofPTH peptide in a blood plasma” are pharmacokinetic parameters known toone skilled in the art. Laursen et al., Eur. J. Endocrinology,135:309-315, 1996. The “concentration vs. time curve” measures theconcentration of PTH peptide in a blood serum of a subject vs. timeafter administration of a dosage of PTH peptide to the subject either byintranasal, intramuscular, subcutaneous, or other parenteral route ofadministration. “C_(max)” is the maximum concentration of PTH peptide inthe blood serum of a subject following a single dosage of PTH peptide tothe subject. “t_(max)” is the time to reach maximum concentration of PTHpeptide in a blood serum of a subject following administration of asingle dosage of PTH peptide to the subject.

While the mechanism of absorption promotion may vary with differentmucosal delivery-enhancing agents of the invention, useful reagents inthis context will not substantially adversely affect the mucosal tissueand will be selected according to the physicochemical characteristics ofthe particular PTH peptide or other active or delivery-enhancing agent.In this context, delivery-enhancing agents that increase penetration orpermeability of mucosal tissues will often result in some alteration ofthe protective permeability barrier of the mucosa. For suchdelivery-enhancing agents to be of value within the invention, it isgenerally desired that any significant changes in permeability of themucosa be reversible within a time frame appropriate to the desiredduration of drug delivery. Furthermore, there should be no substantial,cumulative toxicity, nor any permanent deleterious changes induced inthe barrier properties of the mucosa with long-term use.

Within certain aspects of the invention, absorption-promoting agents forcoordinate administration or combinatorial formulation with PTH peptideof the invention are selected from small hydrophilic molecules,including but not limited to, dimethyl sulfoxide (DMSO),dimethylformamide, ethanol, propylene glycol, and the 2-pyrrolidones.Alternatively, long-chain amphipathic molecules, for example,deacylmethyl sulfoxide, azone, sodium laurylsulfate, oleic acid, and thebile salts, may be employed to enhance mucosal penetration of the PTHpeptide. In additional aspects, surfactants (e.g., polysorbates) areemployed as adjunct compounds, processing agents, or formulationadditives to enhance intranasal delivery of the PTH peptide. Agents suchas DMSO, polyethylene glycol, and ethanol can, if present insufficiently high concentrations in delivery environment (e.g., bypre-administration or incorporation in a therapeutic formulation), enterthe aqueous phase of the mucosa and alter its solubilizing properties,thereby enhancing the partitioning of the PTH peptide from the vehicleinto the mucosa.

Additional mucosal delivery-enhancing agents that are useful within thecoordinate administration and processing methods and combinatorialformulations of the invention include, but are not limited to, mixedmicelles; enamines; nitric oxide donors (e.g.,S-nitroso-N-acetyl-DL-penicillamine, NOR1, NOR4—which are preferablyco-administered with an NO scavenger such as carboxy-PITO or doclofenacsodium); sodium salicylate; glycerol esters of acetoacetic acid (e.g.,glyceryl-1,3-diacetoacetate or1,2-isopropylideneglycerine-3-acetoacetate); and other release-diffusionor intra- or trans-epithelial penetration-promoting agents that arephysiologically compatible for mucosal delivery. Otherabsorption-promoting agents are selected from a variety of carriers,bases and excipients that enhance mucosal delivery, stability, activityor trans-epithelial penetration of the PTH peptide. These include, interalia, cyclodextrins and β-cyclodextrin derivatives (e.g.,2-hydroxypropyl-β-cyclodextrin andheptakis(2,6-di-O-methyl-β-cyclodextrin). These compounds, optionallyconjugated with one or more of the active ingredients and furtheroptionally formulated in an oleaginous base, enhance bioavailability inthe mucosal formulations of the invention. Yet additionalabsorption-enhancing agents adapted for mucosal delivery includemedium-chain fatty acids, including mono- and diglycerides (e.g., sodiumcaprate—extracts of coconut oil, Capmul), and triglycerides (e.g.,amylodextrin, Estaram 299, Miglyol 810).

The mucosal therapeutic and prophylactic compositions of the presentinvention may be supplemented with any suitable penetration-promotingagent that facilitates absorption, diffusion, or penetration of PTHpeptide across mucosal barriers. The penetration promoter may be anypromoter that is pharmaceutically acceptable. Thus, in more detailedaspects of the invention compositions are provided that incorporate oneor more penetration-promoting agents selected from sodium salicylate andsalicylic acid derivatives (acetyl salicylate, choline salicylate,salicylamide, etc.); amino acids and salts thereof (e.g.,monoaminocarboxlic acids such as glycine, alanine, phenylalanine,proline, hydroxyproline, etc.; hydroxyamino acids such as serine; acidicamino acids such as aspartic acid, glutamic acid, etc.; and basic aminoacids such as lysine, etc.—inclusive of their alkali metal or alkalineearth metal salts); and N-acetylamino acids (N-acetylalanine,N-acetylphenylalanine,

N-acetylserine, N-acetylglycine, N-acetyllysine, N-acetylglutamic acid,N-acetylproline, N-acetylhydroxyproline, etc.) and their salts (alkalimetal salts and alkaline earth metal salts). Also provided aspenetration-promoting agents within the methods and compositions of theinvention are substances which are generally used as emulsifiers (e.g.,sodium oleyl phosphate, sodium lauryl phosphate, sodium lauryl sulfate,sodium myristyl sulfate, polyoxyethylene alkyl ethers, polyoxyethylenealkyl esters, etc.), caproic acid, lactic acid, malic acid and citricacid and alkali metal salts thereof, pyrrolidonecarboxylic acids,alkylpyrrolidonecarboxylic acid esters, N-alkylpyrrolidones, prolineacyl esters, and the like.

Within various aspects of the invention, improved nasal mucosal deliveryformulations and methods are provided that allow delivery of PTH peptideand other therapeutic agents within the invention across mucosalbarriers between administration and selected target sites. Certainformulations are specifically adapted for a selected target cell, tissueor organ, or even a particular disease state. In other aspects,formulations and methods provide for efficient, selective endo- ortranscytosis of PTH peptide specifically routed along a definedintracellular or intercellular pathway. Typically, the PTH peptide isefficiently loaded at effective concentration levels in a carrier orother delivery vehicle, and is delivered and maintained in a stabilizedform, e.g., at the nasal mucosa and/or during passage throughintracellular compartments and membranes to a remote target site fordrug action (e.g., the blood stream or a defined tissue, organ, orextracellular compartment). The PTH peptide may be provided in adelivery vehicle or otherwise modified (e.g., in the form of a prodrug),wherein release or activation of the PTH peptide is triggered by aphysiological stimulus (e.g. pH change, lysosomal enzymes, etc.). Often,the PTH peptide is pharmacologically inactive until it reaches itstarget site for activity. In most cases, the PTH peptide and otherformulation components are non-toxic and non-immunogenic. In thiscontext, carriers and other formulation components are generallyselected for their ability to be rapidly degraded and excreted underphysiological conditions. At the same time, formulations are chemicallyand physically stable in dosage form for effective storage.

Included within the definition of biologically active peptides andproteins for use within the invention are natural or synthetic,therapeutically or prophylactically active, peptides (comprised of twoor more covalently linked amino acids), proteins, peptide or proteinfragments, peptide or protein analogs, and chemically modifiedderivatives or salts of active peptides or proteins. A wide variety ofuseful analogs and mimetics of PTH peptide are contemplated for usewithin the invention and can be produced and tested for biologicalactivity according to known methods. Often, the peptides or proteins ofPTH peptide or other biologically active peptides or proteins for usewithin the invention are muteins that are readily obtainable by partialsubstitution, addition, or deletion of amino acids within a naturallyoccurring or native (e.g., wild-type, naturally occurring mutant, orallelic variant) peptide or protein sequence. Additionally, biologicallyactive fragments of native peptides or proteins are included. Suchmutant derivatives and fragments substantially retain the desiredbiological activity of the native peptide or proteins. In the case ofpeptides or proteins having carbohydrate chains, biologically activevariants marked by alterations in these carbohydrate species are alsoincluded within the invention.

As used herein, the term “conservative amino acid substitution” refersto the general interchangeability of amino acid residues having similarside chains. For example, a commonly interchangeable group of aminoacids having aliphatic side chains is alanine, valine, leucine, andisoleucine; a group of amino acids having aliphatic-hydroxyl side chainsis serine and threonine; a group of amino acids having amide-containingside chains is asparagine and glutamine; a group of amino acids havingaromatic side chains is phenylalanine, tyrosine, and tryptophan; a groupof amino acids having basic side chains is lysine, arginine, andhistidine; and a group of amino acids having sulfur-containing sidechains is cysteine and methionine. Examples of conservativesubstitutions include the substitution of a non-polar (hydrophobic)residue such as isoleucine, valine, leucine or methionine for another.Likewise, the present invention contemplates the substitution of a polar(hydrophilic) residue such as between arginine and lysine, betweenglutamine and asparagine, and between threonine and serine.Additionally, the substitution of a basic residue such as lysine,arginine or histidine for another or the substitution of an acidicresidue such as aspartic acid or glutamic acid for another is alsocontemplated. Exemplary conservative amino acids substitution groupsare: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine,alanine-valine, and asparagine-glutamine. By aligning a peptide orprotein analog optimally with a corresponding native peptide or protein,and by using appropriate assays, e.g., adhesion protein or receptorbinding assays, to determine a selected biological activity, one canreadily identify operable peptide and protein analogs for use within themethods and compositions of the invention. Operable peptide and proteinanalogs are typically specifically immunoreactive with antibodies raisedto the corresponding native peptide or protein.

An approach for stabilizing solid protein formulations of the inventionis to increase the physical stability of purified, e.g., lyophilized,protein. This will inhibit aggregation via hydrophobic interactions aswell as via covalent pathways that may increase as proteins unfold.Stabilizing formulations in this context often include polymer-basedformulations, for example a biodegradable hydrogel formulation/deliverysystem. As noted above, the critical role of water in protein structure,function, and stability is well known. Typically, proteins arerelatively stable in the solid state with bulk water removed. However,solid therapeutic protein formulations may become hydrated upon storageat elevated humidities or during delivery from a sustained releasecomposition or device. The stability of proteins generally drops withincreasing hydration. Water can also play a significant role in solidprotein aggregation, for example, by increasing protein flexibilityresulting in enhanced accessibility of reactive groups, by providing amobile phase for reactants, and by serving as a reactant in severaldeleterious processes such as beta-elimination and hydrolysis.

Protein preparations containing between about 6% to 28% water are themost unstable. Below this level, the mobility of bound water and proteininternal motions are low. Above this level, water mobility and proteinmotions approach those of full hydration. Up to a point, increasedsusceptibility toward solid-phase aggregation with increasing hydrationhas been observed in several systems. However, at higher water content,less aggregation is observed because of the dilution effect.

In accordance with these principles, an effective method for stabilizingpeptides and proteins against solid-state aggregation for mucosaldelivery is to control the water content in a solid formulation andmaintain the water activity in the formulation at optimal levels. Thislevel depends on the nature of the protein, but in general, proteinsmaintained below their “monolayer” water coverage will exhibit superiorsolid-state stability.

A variety of additives, diluents, bases and delivery vehicles areprovided within the invention that effectively control water content toenhance protein stability. These reagents and carrier materialseffective as anti-aggregation agents in this sense include, for example,polymers of various functionalities, such as polyethylene glycol,dextran, diethylaminoethyl dextran, and carboxymethyl cellulose, whichsignificantly increase the stability and reduce the solid-phaseaggregation of peptides and proteins admixed therewith or linkedthereto. In some instances, the activity or physical stability ofproteins can also be enhanced by various additives to aqueous solutionsof the peptide or protein drugs. For example, additives, such as polyols(including sugars), amino acids, proteins such as collagen and gelatin,and various salts may be used.

Certain additives, in particular sugars and other polyols, also impartsignificant physical stability to dry, e.g., lyophilized proteins. Theseadditives can also be used within the invention to protect the proteinsagainst aggregation not only during lyophilization but also duringstorage in the dry state. For example sucrose and Ficoll 70 (a polymerwith sucrose units) exhibit significant protection against peptide orprotein aggregation during solid-phase incubation under variousconditions. These additives may also enhance the stability of solidproteins embedded within polymer matrices.

Yet additional additives, for example sucrose, stabilize proteinsagainst solid-state aggregation in humid atmospheres at elevatedtemperatures, as may occur in certain sustained-release formulations ofthe invention. Proteins such as gelatin and collagen also serve asstabilizing or bulking agents to reduce denaturation and aggregation ofunstable proteins in this context. These additives can be incorporatedinto polymeric melt processes and compositions within the invention. Forexample, polypeptide microparticles can be prepared by simplylyophilizing or spray drying a solution containing various stabilizingadditives described above. Sustained release of unaggregated peptidesand proteins can thereby be obtained over an extended period of time.

Various additional preparative components and methods, as well asspecific formulation additives, are provided herein which yieldformulations for mucosal delivery of aggregation-prone peptides andproteins, wherein the peptide or protein is stabilized in asubstantially pure, unaggregated form using a solubilization agent. Arange of components and additives are contemplated for use within thesemethods and formulations. Exemplary of these solubilization agents arecyclodextrins (CDs), which selectively bind hydrophobic side chains ofpolypeptides. These CDs have been found to bind to hydrophobic patchesof proteins in a manner that significantly inhibits aggregation. Thisinhibition is selective with respect to both the CD and the proteininvolved. Such selective inhibition of protein aggregation providesadditional advantages within the intranasal delivery methods andcompositions of the invention. Additional agents for use in this contextinclude CD dimers, trimers and tetramers with varying geometriescontrolled by the linkers that specifically block aggregation ofpeptides and protein. Yet solubilization agents and methods forincorporation within the invention involve the use of peptides andpeptide mimetics to selectively block protein-protein interactions. Inone aspect, the specific binding of hydrophobic side chains reported forCD multimers is extended to proteins via the use of peptides and peptidemimetics that similarly block protein aggregation. A wide range ofsuitable methods and anti-aggregation agents are available forincorporation within the compositions and procedures of the invention.

To improve the transport characteristics of biologically active agents(including PTH peptide, other active peptides and proteins, andmacromolecular and small molecule drugs) for enhanced delivery acrosshydrophobic mucosal membrane barriers, the invention also providestechniques and reagents for charge modification of selected biologicallyactive agents or delivery-enhancing agents described herein. In thisregard, the relative permeabilities of macromolecules is generally berelated to their partition coefficients. The degree of ionization ofmolecules, which is dependent on the pK_(a). of the molecule and the pHat the mucosal membrane surface, also affects permeability of themolecules. Permeation and partitioning of biologically active agents,including PTH peptide and analogs of the invention, for mucosal deliverymay be facilitated by charge alteration or charge spreading of theactive agent or permeabilizing agent, which is achieved, for example, byalteration of charged functional groups, by modifying the pH of thedelivery vehicle or solution in which the active agent is delivered, orby coordinate administration of a charge- or pH-altering reagent withthe active agent.

Consistent with these general teachings, mucosal delivery of chargedmacromolecular species, including PTH peptide and other biologicallyactive peptides and proteins, within the methods and compositions of theinvention is substantially improved when the active agent is deliveredto the mucosal surface in a substantially un-ionized, or neutral,electrical charge state.

Certain PTH peptide and other biologically active peptide and proteincomponents of mucosal formulations for use within the invention will becharge modified to yield an increase in the positive charge density ofthe peptide or protein. These modifications extend also to cationizationof peptide and protein conjugates, carriers and other delivery formsdisclosed herein. Cationization offers a convenient means of alteringthe biodistribution and transport properties of proteins andmacromolecules within the invention. Cationization is undertaken in amanner that substantially preserves the biological activity of theactive agent and limits potentially adverse side effects, includingtissue damage and toxicity.

Effective delivery of biotherapeutic agents via intranasaladministration must take into account the decreased drug transport rateacross the protective mucus lining of the nasal mucosa, in addition todrug loss due to binding to glycoproteins of the mucus layer. Normalmucus is a viscoelastic, gel-like substance consisting of water,electrolytes, mucins, macromolecules, and sloughed epithelial cells. Itserves primarily as a cytoprotective and lubricative covering for theunderlying mucosal tissues. Mucus is secreted by randomly distributedsecretory cells located in the nasal epithelium and in other mucosalepithelia. The structural unit of mucus is mucin. This glycoprotein ismainly responsible for the viscoelastic nature of mucus, although othermacromolecules may also contribute to this property. In airway mucus,such macromolecules include locally produced secretory IgA, IgM, IgE,lysozyme, and bronchotransferrin, which also play an important role inhost defense mechanisms.

The coordinate administration methods of the instant inventionoptionally incorporate effective mucolytic or mucus-clearing agents,which serve to degrade, thin or clear mucus from intranasal mucosalsurfaces to facilitate absorption of intranasally administeredbiotherapeutic agents. Within these methods, a mucolytic ormucus-clearing agent is coordinately administered as an adjunct compoundto enhance intranasal delivery of the biologically active agent.Alternatively, an effective amount of a mucolytic or mucus-clearingagent is incorporated as a processing agent within a multi-processingmethod of the invention, or as an additive within a combinatorialformulation of the invention, to provide an improved formulation thatenhances intranasal delivery of biotherapeutic compounds by reducing thebarrier effects of intranasal mucus.

A variety of mucolytic or mucus-clearing agents are available forincorporation within the methods and compositions of the invention.Based on their mechanisms of action, mucolytic and mucus clearing agentscan often be classified into the following groups: proteases (e.g.,pronase, papain) that cleave the protein core of mucin glycoproteins;sulfhydryl compounds that split mucoprotein disulfide linkages; anddetergents (e.g., Triton X-100, Tween 20) that break non-covalent bondswithin the mucus. Additional compounds in this context include, but arenot limited to, bile salts and surfactants, for example, sodiumdeoxycholate, sodium taurodeoxycholate, sodium glycocholate, andlysophosphatidylcholine.

The effectiveness of bile salts in causing structural breakdown of mucusis in the order deoxycholate>taurocholate>glycocholate. Other effectiveagents that reduce mucus viscosity or adhesion to enhance intranasaldelivery according to the methods of the invention include, e.g.,short-chain fatty acids, and mucolytic agents that work by chelation,such as N-acylcollagen peptides, bile acids, and saponins (the latterfunction in part by chelating Ca²⁺ and/or Mg²⁺ which play an importantrole in maintaining mucus layer structure).

Additional mucolytic agents for use within the methods and compositionsof the invention include N-acetyl-L-cysteine (ACS), a potent mucolyticagent that reduces both the viscosity and adherence of bronchopulmonarymucus and is reported to modestly increase nasal bioavailability ofhuman growth hormone in anesthetized rats (from 7.5 to 12.2%). These andother mucolytic or mucus-clearing agents are contacted with the nasalmucosa, typically in a concentration range of about 0.2 to 20 mM,coordinately with administration of the biologically active agent, toreduce the polar viscosity and/or elasticity of intranasal mucus.

Still other mucolytic or mucus-clearing agents may be selected from arange of glycosidase enzymes, which are able to cleave glycosidic bondswithin the mucus glycoprotein. α-amylase and β-amylase arerepresentative of this class of enzymes, although their mucolytic effectmay be limited. In contrast, bacterial glycosidases which allow thesemicroorganisms to permeate mucus layers of their hosts.

For combinatorial use with most biologically active agents within theinvention, including peptide and protein therapeutics, non-ionogenicdetergents are generally also useful as mucolytic or mucus-clearingagents. These agents typically will not modify or substantially impairthe activity of therapeutic polypeptides.

Because the self-cleaning capacity of certain mucosal tissues (e.g.,nasal mucosal tissues) by mucociliary clearance is necessary as aprotective function (e.g., to remove dust, allergens, and bacteria), ithas been generally considered that this function should not besubstantially impaired by mucosal medications. Mucociliary transport inthe respiratory tract is a particularly important defense mechanismagainst infections. To achieve this function, ciliary beating in thenasal and airway passages moves a layer of mucus along the mucosa toremoving inhaled particles and microorganisms.

Ciliostatic agents find use within the methods and compositions of theinvention to increase the residence time of mucosally (e.g.,intranasally) administered PTH peptide, analogs and mimetics, and otherbiologically active agents disclosed herein. In particular, the deliverythese agents within the methods and compositions of the invention issignificantly enhanced in certain aspects by the coordinateadministration or combinatorial formulation of one or more ciliostaticagents that function to reversibly inhibit ciliary activity of mucosalcells, to provide for a temporary, reversible increase in the residencetime of the mucosally administered active agent(s). For use within theseaspects of the invention, the foregoing ciliostatic factors, eitherspecific or indirect in their activity, are all candidates forsuccessful employment as ciliostatic agents in appropriate amounts(depending on concentration, duration and mode of delivery) such thatthey yield a transient (i.e., reversible) reduction or cessation ofmucociliary clearance at a mucosal site of administration to enhancedelivery of PTH peptide, analogs and mimetics, and other biologicallyactive agents disclosed herein, without unacceptable adverse sideeffects.

Within more detailed aspects, a specific ciliostatic factor is employedin a combined formulation or coordinate administration protocol with oneor more PTH peptide proteins, analogs and mimetics, and/or otherbiologically active agents disclosed herein. Various bacterialciliostatic factors isolated and characterized in the literature may beemployed within these embodiments of the invention. Ciliostatic factorsfrom the bacterium Pseudomonas aeruginosa include a phenazinederivative, a pyo compound (2-alkyl-4-hydroxyquinolines), and arhamnolipid (also known as a hemolysin). The pyo compound producedciliostasis at concentrations of 50 μg/ml and without obviousultrastructural lesions. The phenazine derivative also inhibited ciliarymotility but caused some membrane disruption, although at substantiallygreater concentrations of 400 μg/ml. Limited exposure of trachealexplants to the rhamnolipid resulted in ciliostasis, which wasassociated with altered ciliary membranes. More extensive exposure torhamnolipid was associated with removal of dynein arms from axonemes.

Within more detailed aspects of the invention, one or more membranepenetration-enhancing agents may be employed within a mucosal deliverymethod or formulation of the invention to enhance mucosal delivery ofPTH peptide analogs and mimetics, and other biologically active agentsdisclosed herein. Membrane penetration enhancing agents in this contextcan be selected from: (i) a surfactant, (ii) a bile salt, (iii) aphospholipid additive, mixed micelle, liposome, or carrier, (iv) analcohol, (v) an enamine, (vi) an NO donor compound, (vii) a long-chainamphipathic molecule (viii) a small hydrophobic penetration enhancer;(ix) sodium or a salicylic acid derivative; (x) a glycerol ester ofacetoacetic acid (xi) a cyclodextrin or beta-cyclodextrin derivative,(xii) a medium-chain fatty acid, (xiii) a chelating agent, (xiv) anamino acid or salt thereof, (xv) an N-acetylamino acid or salt thereof,(xvi) an enzyme degradative to a selected membrane component, (xvii) aninhibitor of fatty acid synthesis, or (xviii) an inhibitor ofcholesterol synthesis; or (xix) any combination of the membranepenetration enhancing agents recited in (i)-(xviii).

Certain surface-active agents are readily incorporated within themucosal delivery formulations and methods of the invention as mucosalabsorption enhancing agents. These agents, which may be coordinatelyadministered or combinatorially formulated with PTH peptide proteins,analogs and mimetics, and other biologically active agents disclosedherein, may be selected from a broad assemblage of known surfactants.Surfactants, which generally fall into three classes: (1) nonionicpolyoxyethylene ethers; (2) bile salts such as sodium glycocholate (SGC)and deoxycholate (DOC); and (3) derivatives of fusidic acid such assodium taurodihydrofusidate (STDHF). The mechanisms of action of thesevarious classes of surface-active agents typically includesolubilization of the biologically active agent. For proteins andpeptides which often form aggregates, the surface active properties ofthese absorption promoters can allow interactions with proteins suchthat smaller units such as surfactant coated monomers may be morereadily maintained in solution. Examples of other surface-active agentsare L-α-Phosphatidylcholine Didecanoyl (DDPC) polysorbate 80 andpolysorbate 20. These monomers are presumably more transportable unitsthan aggregates. A second potential mechanism is the protection of thepeptide or protein from proteolytic degradation by proteases in themucosal environment. Both bile salts and some fusidic acid derivativesreportedly inhibit proteolytic degradation of proteins by nasalhomogenates at concentrations less than or equivalent to those requiredto enhance protein absorption. This protease inhibition may beespecially important for peptides with short biological half-lives.

The present invention provides pharmaceutical composition that containsone or more PTH peptides, analogs or mimetics, and/or other biologicallyactive agents in combination with mucosal delivery enhancing agentsdisclosed herein formulated in a pharmaceutical preparation for mucosaldelivery.

The permeabilizing agent reversibly enhances mucosal epithelialparacellular transport, typically by modulating epithelial junctionalstructure and/or physiology at a mucosal epithelial surface in thesubject. This effect typically involves inhibition by the permeabilizingagent of homotypic or heterotypic binding between epithelial membraneadhesive proteins of neighboring epithelial cells. Target proteins forthis blockade of homotypic or heterotypic binding can be selected fromvarious related junctional adhesion molecules (JAMs), occludins, orclaudins. Examples of this are antibodies, antibody fragments orsingle-chain antibodies that bind to the extracellular domains of theseproteins.

In yet additional detailed embodiments, the invention providespermeabilizing peptides and peptide analogs and mimetics for enhancingmucosal epithelial paracellular transport. The subject peptides andpeptide analogs and mimetics typically work within the compositions andmethods of the invention by modulating epithelial junctional structureand/or physiology in a mammalian subject. In certain embodiments, thepeptides and peptide analogs and mimetics effectively inhibit homotypicand/or heterotypic binding of an epithelial membrane adhesive proteinselected from a junctional adhesion molecule (JAM), occludin, orclaudin.

One such agent that has been extensively studied is the bacterial toxinfrom Vibrio cholerae known as the “zonula occludens toxin” (ZOT). Thistoxin mediates increased intestinal mucosal permeability and causesdisease symptoms including diarrhea in infected subjects. Fasano et al.,Proc. Nat. Acad. Sci., U.S.A. 8:5242-5246, 1991. When tested on rabbitileal mucosa, ZOT increased the intestinal permeability by modulatingthe structure of intercellular tight junctions. More recently, it hasbeen found that ZOT is capable of reversibly opening tight junctions inthe intestinal mucosa. It has also been reported that ZOT is capable ofreversibly opening tight junctions in the nasal mucosa. U.S. Pat. No.5,908,825.

Within the methods and compositions of the invention, ZOT, as well asvarious analogs and mimetics of ZOT that function as agonists orantagonists of ZOT activity, are useful for enhancing intranasaldelivery of biologically active agents—by increasing paracellularabsorption into and across the nasal mucosa. In this context, ZOTtypically acts by causing a structural reorganization of tight junctionsmarked by altered localization of the junctional protein ZO1. Withinthese aspects of the invention, ZOT is coordinately administered orcombinatorially formulated with the biologically active agent in aneffective amount to yield significantly enhanced absorption of theactive agent, by reversibly increasing nasal mucosal permeabilitywithout substantial adverse side effects.

The compositions and delivery methods of the invention optionallyincorporate a selective transport-enhancing agent that facilitatestransport of one or more biologically active agents. Thesetransport-enhancing agents may be employed in a combinatorialformulation or coordinate administration protocol with one or more ofthe PTH peptides, analogs and mimetics disclosed herein, to coordinatelyenhance delivery of one or more additional biologically active agent(s)across mucosal transport barriers, to enhance mucosal delivery of theactive agent(s) to reach a target tissue or compartment in the subject(e.g., the mucosal epithelium, liver, CNS tissue or fluid, or bloodplasma). Alternatively, the transport-enhancing agents may be employedin a combinatorial formulation or coordinate administration protocol todirectly enhance mucosal delivery of one or more of the PTH peptides,analogs and mimetics, with or without enhanced delivery of an additionalbiologically active agent.

Exemplary selective transport-enhancing agents for use within thisaspect of the invention include, but are not limited to, glycosides,sugar-containing molecules, and binding agents such as lectin bindingagents, which are known to interact specifically with epithelialtransport barrier components. For example, specific “bioadhesive”ligands, including various plant and bacterial lectins, which bind tocell surface sugar moieties by receptor-mediated interactions can beemployed as carriers or conjugated transport mediators for enhancingmucosal, e.g., nasal delivery of biologically active agents within theinvention. Certain bioadhesive ligands for use within the invention willmediate transmission of biological signals to epithelial target cellsthat trigger selective uptake of the adhesive ligand by specializedcellular transport processes (endocytosis or transcytosis). Thesetransport mediators can therefore be employed as a “carrier system” tostimulate or direct selective uptake of one or more PTH peptides,analogs and mimetics, and other biologically active agent(s) into and/orthrough mucosal epithelia. These and other selective transport-enhancingagents significantly enhance mucosal delivery of macromolecularbiopharmaceuticals (particularly peptides, proteins, oligonucleotidesand polynucleotide vectors) within the invention. Lectins are plantproteins that bind to specific sugars found on the surface ofglycoproteins and glycolipids of eukaryotic cells. Concentratedsolutions of lectins have a ‘mucotractive’ effect, and various studieshave demonstrated rapid receptor mediated endocytocis (RME) of lectinsand lectin conjugates (e.g., concanavalin A conjugated with colloidalgold particles) across mucosal surfaces. Additional studies havereported that the uptake mechanisms for lectins can be utilized forintestinal drug targeting in vivo. In certain of these studies,polystyrene nanoparticles (500 nm) were covalently coupled to tomatolectin and reported yielded improved systemic uptake after oraladministration to rats.

In addition to plant lectins, microbial adhesion and invasion factorsprovide a rich source of candidates for use as adhesive/selectivetransport carriers within the mucosal delivery methods and compositionsof the invention. Two components are necessary for bacterial adherenceprocesses, a bacterial ‘adhesin’ (adherence or colonization factor) anda receptor on the host cell surface. Bacteria causing mucosal infectionsneed to penetrate the mucus layer before attaching themselves to theepithelial surface. This attachment is usually mediated by bacterialfimbriae or pilus structures, although other cell surface components mayalso take part in the process. Adherent bacteria colonize mucosalepithelia by multiplication and initiation of a series of biochemicalreactions inside the target cell through signal transduction mechanisms(with or without the help of toxins). Associated with these invasivemechanisms, a wide diversity of bioadhesive proteins (e.g., invasin,internalin) originally produced by various bacteria and viruses areknown. These allow for extracellular attachment of such microorganismswith an impressive selectivity for host species and even particulartarget tissues. Signals transmitted by such receptor-ligand interactionstrigger the transport of intact, living microorganisms into, andeventually through, epithelial cells by endo- and transcytoticprocesses. Such naturally occurring phenomena may be harnessed (e.g., bycomplexing biologically active agents such as PTH peptides with anadhesin) according to the teachings herein for enhanced delivery ofbiologically active compounds into or across mucosal epithelia and/or toother designated target sites of drug action.

Various bacterial and plant toxins that bind epithelial surfaces in aspecific, lectin-like manner are also useful within the methods andcompositions of the invention. For example, diptheria toxin (DT) entershost cells rapidly by RME. Likewise, the B subunit of the E. coli heatlabile toxin binds to the brush border of intestinal epithelial cells ina highly specific, lectin-like manner. Uptake of this toxin andtranscytosis to the basolateral side of the enterocytes has beenreported in vivo and in vitro. Other researches have expressed thetransmembrane domain of diphtheria toxin in E. coli as a maltose-bindingfusion protein and coupled it chemically to high-Mw poly-L-lysine. Theresulting complex was successfully used to mediate internalization of areporter gene in vitro. In addition to these examples, Staphylococcusaureus produces a set of proteins (e.g., staphylococcal enterotoxin A(SEA), SEB, toxic shock syndrome toxin 1 (TSST-1) which act both assuperantigens and toxins. Studies relating to these proteins havereported dose-dependent, facilitated transcytosis of SEB and TSST-I inCaco-2 cells.

Viral haemagglutinins comprise another type of transport agent tofacilitate mucosal delivery of biologically active agents within themethods and compositions of the invention. The initial step in manyviral infections is the binding of surface proteins (haemagglutinins) tomucosal cells. These binding proteins have been identified for mostviruses, including rotaviruses, varicella zoster virus, semliki forestvirus, adenoviruses, potato leafroll virus, and reovirus. These andother exemplary viral hemagglutinins can be employed in a combinatorialformulation (e.g., a mixture or conjugate formulation) or coordinateadministration protocol with one or more of the PTH peptide, analogs andmimetics disclosed herein, to coordinately enhance mucosal delivery ofone or more additional biologically active agent(s). Alternatively,viral hemagglutinins can be employed in a combinatorial formulation orcoordinate administration protocol to directly enhance mucosal deliveryof one or more of the PTH peptide proteins, analogs and mimetics, withor without enhanced delivery of an additional biologically active agent.

A variety of endogenous, selective transport-mediating factors are alsoavailable for use within the invention. Mammalian cells have developedan assortment of mechanisms to facilitate the internalization ofspecific substrates and target these to defined compartments.Collectively, these processes of membrane deformations are termed‘endocytosis’ and comprise phagocytosis, pinocytosis, receptor-mediatedendocytosis (clathrin-mediated RME), and potocytosis(non-clathrin-mediated RME). RME is a highly specific cellular biologicprocess by which, as its name implies, various ligands bind to cellsurface receptors and are subsequently internalized and traffickedwithin the cell. In many cells the process of endocytosis is so activethat the entire membrane surface is internalized and replaced in lessthan a half hour. Two classes of receptors are proposed based on theirorientation in the cell membrane; the amino terminus of Type I receptorsis located on the extracellular side of the membrane, whereas Type IIreceptors have this same protein tail in the intracellular milieu.

Still other embodiments of the invention utilize transferrin as acarrier or stimulant of RME of mucosally delivered biologically activeagents. Transferrin, an 80 kDa iron-transporting glycoprotein, isefficiently taken up into cells by RME. Transferrin receptors are foundon the surface of most proliferating cells, in elevated numbers onerythroblasts and on many kinds of tumors. The transcytosis oftransferrin (TO and transferrin conjugates is reportedly enhanced in thepresence of Brefeldin A (BFA), a fungal metabolite. In other studies,BFA treatment has been reported to rapidly increase apical endocytosisof both ricin and HRP in MDCK cells. Thus, BFA and other agents thatstimulate receptor-mediated transport can be employed within the methodsof the invention as combinatorially formulated (e.g., conjugated) and/orcoordinately administered agents to enhance receptor-mediated transportof biologically active agents, including PTH peptide proteins, analogsand mimetics.

In certain aspects of the invention, the combinatorial formulationsand/or coordinate administration methods herein incorporate an effectiveamount of peptides and proteins which may adhere to charged glassthereby reducing the effective concentration in the container. Silanizedcontainers, for example, silanized glass containers, are used to storethe finished product to reduce adsorption of the polypeptide or proteinto a glass container.

In yet additional aspects of the invention, a kit for treatment of amammalian subject comprises a stable pharmaceutical composition of oneor more PTH peptide compound(s) formulated for mucosal delivery to themammalian subject wherein the composition is effective for treating orpreventing osteoporosis or osteopenia. The kit further comprises apharmaceutical reagent vial to contain the one or more PTH peptidecompounds. The pharmaceutical reagent vial is composed of pharmaceuticalgrade polymer, glass or other suitable material. The pharmaceuticalreagent vial is, for example, a silanized glass vial. The kit furthercomprises an aperture for delivery of the composition to a nasal mucosalsurface of the subject. The delivery aperture is composed of apharmaceutical grade polymer, glass or other suitable material. Thedelivery aperture is, for example, a silanized glass.

A silanization technique combines a special cleaning technique for thesurfaces to be silanized with a silanization process at low pressure.The silane is in the gas phase and at an enhanced temperature of thesurfaces to be silanized. The method provides reproducible surfaces withstable, homogeneous and functional silane layers having characteristicsof a monolayer. The silanized surfaces prevent binding to the glass ofpolypeptides or mucosal delivery enhancing agents of the presentinvention.

The procedure is useful to prepare silanized pharmaceutical reagentvials to hold PTH peptide compositions of the present invention. Glasstrays are cleaned by rinsing with double distilled water (ddH₂O) beforeusing. The silane tray is then be rinsed with 95% EtOH, and the acetonetray is rinsed with acetone. Pharmaceutical reagent vials are sonicatedin acetone for 10 minutes. After the acetone sonication, reagent vialsare washed in ddH₂O tray at least twice. Reagent vials are sonicated in0.1M NaOH for 10 minutes. While the reagent vials are sonicating inNaOH, the silane solution is made under a hood. (Silane solution: 800 mLof 95% ethanol; 96 L of glacial acetic acid; 25 mL ofglycidoxypropyltrimethoxy silane).

After the NaOH sonication, reagent vials are washed in ddH₂O tray atleast twice. The reagent vials are sonicated in silane solution for 3 to5 minutes. The reagent vials are washed in 100% EtOH tray. The reagentvials are dried with prepurified N₂ gas and stored in a 100° C. oven forat least 2 hours before using.

In certain aspects of the invention, the combinatorial formulationsand/or coordinate administration methods herein incorporate an effectiveamount of a nontoxic bioadhesive as an adjunct compound or carrier toenhance mucosal delivery of one or more biologically active agent(s).Bioadhesive agents in this context exhibit general or specific adhesionto one or more components or surfaces of the targeted mucosa. Thebioadhesive maintains a desired concentration gradient of thebiologically active agent into or across the mucosa to ensurepenetration of even large molecules (e.g., peptides and proteins) intoor through the mucosal epithelium. Typically, employment of abioadhesive within the methods and compositions of the invention yieldsa two- to five-fold, often a five- to ten-fold increase in permeabilityfor peptides and proteins into or through the mucosal epithelium. Thisenhancement of epithelial permeation often permits effectivetransmucosal delivery of large macromolecules, for example to the basalportion of the nasal epithelium or into the adjacent extracellularcompartments or a blood plasma or CNS tissue or fluid.

This enhanced delivery provides for greatly improved effectiveness ofdelivery of bioactive peptides, proteins and other macromoleculartherapeutic species. These results will depend in part on thehydrophilicity of the compound, whereby greater penetration will beachieved with hydrophilic species compared to water insoluble compounds.In addition to these effects, employment of bioadhesives to enhance drugpersistence at the mucosal surface can elicit a reservoir mechanism forprotracted drug delivery, whereby compounds not only penetrate acrossthe mucosal tissue but also back-diffuse toward the mucosal surface oncethe material at the surface is depleted.

A variety of suitable bioadhesives are disclosed in the art for oraladministration, U.S. Pat. Nos. 3,972,995; 4,259,314; 4,680,323;4,740,365; 4,573,996; 4,292,299; 4,715,369; 4,876,092; 4,855,142;4,250,163; 4,226,848; 4,948,580; U.S. Pat. Reissue No. 33,093, herebyincorporated by reference, which find use within the novel methods andcompositions of the invention. The potential of various bioadhesivepolymers as a mucosal, e.g., nasal, delivery platform within the methodsand compositions of the invention can be readily assessed by determiningtheir ability to retain and release PTH peptide, as well as by theircapacity to interact with the mucosal surfaces following incorporationof the active agent therein. In addition, well known methods will beapplied to determine the biocompatibility of selected polymers with thetissue at the site of mucosal administration. When the target mucosa iscovered by mucus (i.e., in the absence of mucolytic or mucus-clearingtreatment), it can serve as a connecting link to the underlying mucosalepithelium. Therefore, the term “bioadhesive” as used herein also coversmucoadhesive compounds useful for enhancing mucosal delivery ofbiologically active agents within the invention. However, adhesivecontact to mucosal tissue mediated through adhesion to a mucus gel layermay be limited by incomplete or transient attachment between the mucuslayer and the underlying tissue, particularly at nasal surfaces whererapid mucus clearance occurs. In this regard, mucin glycoproteins arecontinuously secreted and, immediately after their release from cells orglands, form a viscoelastic gel. The luminal surface of the adherent gellayer, however, is continuously eroded by mechanical, enzymatic and/orciliary action. Where such activities are more prominent or where longeradhesion times are desired, the coordinate administration methods andcombinatorial formulation methods of the invention may furtherincorporate mucolytic and/or ciliostatic methods or agents as disclosedherein above.

Typically, mucoadhesive polymers for use within the invention arenatural or synthetic macromolecules which adhere to wet mucosal tissuesurfaces by complex, but non-specific, mechanisms. In addition to thesemucoadhesive polymers, the invention also provides methods andcompositions incorporating bioadhesives that adhere directly to a cellsurface, rather than to mucus, by means of specific, includingreceptor-mediated, interactions. One example of bioadhesives thatfunction in this specific manner is the group of compounds known aslectins. These are glycoproteins with an ability to specificallyrecognize and bind to sugar molecules, e.g., glycoproteins orglycolipids, which form part of intranasal epithelial cell membranes andcan be considered as “lectin receptors.”

In certain aspects of the invention, bioadhesive materials for enhancingintranasal delivery of biologically active agents comprise a matrix of ahydrophilic, e.g., water soluble or swellable, polymer or a mixture ofpolymers that can adhere to a wet mucous surface. These adhesives may beformulated as ointments, hydrogels (see above) thin films, and otherapplication forms. Often, these adhesives have the biologically activeagent mixed therewith to effectuate slow release or local delivery ofthe active agent. Some are formulated with additional ingredients tofacilitate penetration of the active agent through the nasal mucosa,e.g., into the circulatory system of the individual.

Various polymers, both natural and synthetic ones, show significantbinding to mucus and/or mucosal epithelial surfaces under physiologicalconditions. The strength of this interaction can readily be measured bymechanical peel or shear tests. When applied to a humid mucosal surface,many dry materials will spontaneously adhere, at least slightly. Aftersuch an initial contact, some hydrophilic materials start to attractwater by adsorption, swelling or capillary forces, and if this water isabsorbed from the underlying substrate or from the polymer-tissueinterface, the adhesion may be sufficient to achieve the goal ofenhancing mucosal absorption of biologically active agents. Such‘adhesion by hydration’ can be quite strong, but formulations adapted toemploy this mechanism must account for swelling which continues as thedosage transforms into a hydrated mucilage. This is projected for manyhydrocolloids useful within the invention, especially somecellulose-derivatives, which are generally non-adhesive when applied inpre-hydrated state. Nevertheless, bioadhesive drug delivery systems formucosal administration are effective within the invention when suchmaterials are applied in the form of a dry polymeric powder,microsphere, or film-type delivery form.

Other polymers adhere to mucosal surfaces not only when applied in dry,but also in fully hydrated state, and in the presence of excess amountsof water. The selection of a mucoadhesive thus requires dueconsideration of the conditions, physiological as well asphysico-chemical, under which the contact to the tissue will be formedand maintained. In particular, the amount of water or humidity usuallypresent at the intended site of adhesion, and the prevailing pH, areknown to largely affect the mucoadhesive binding strength of differentpolymers.

Several polymeric bioadhesive drug delivery systems have been fabricatedand studied in the past 20 years, not always with success. A variety ofsuch carriers are, however, currently used in clinical applicationsinvolving dental, orthopedic, ophthalmological, and surgical uses. Forexample, acrylic-based hydrogels have been used extensively forbioadhesive devices. Acrylic-based hydrogels are well suited forbioadhesion due to their flexibility and nonabrasive characteristics inthe partially swollen state, which reduce damage-causing attrition tothe tissues in contact. Furthermore, their high permeability in theswollen state allows unreacted monomer, un-crosslinked polymer chains,and the initiator to be washed out of the matrix after polymerization,which is an important feature for selection of bioadhesive materials foruse within the invention. Acrylic-based polymer devices exhibit veryhigh adhesive bond strength. For controlled mucosal delivery of peptideand protein drugs, the methods and compositions of the inventionoptionally include the use of carriers, e.g., polymeric deliveryvehicles that function in part to shield the biologically active agentfrom proteolytic breakdown, while at the same time providing forenhanced penetration of the peptide or protein into or through the nasalmucosa. In this context, bioadhesive polymers have demonstratedconsiderable potential for enhancing oral drug delivery. As an example,the bioavailability of 9-desglycinamide, 8-arginine vasopressin (DGAVP)intraduodenally administered to rats together with a 1% (w/v) salinedispersion of the mucoadhesive poly(acrylic acid) derivativepolycarbophil, was 3-5-fold increased compared to an aqueous solution ofthe peptide drug without this polymer.

Mucoadhesive polymers of the poly(acrylic acid)-type are potentinhibitors of some intestinal proteases. The mechanism of enzymeinhibition is explained by the strong affinity of this class of polymersfor divalent cations, such as calcium or zinc, which are essentialcofactors of metallo-proteinases, such as trypsin and chymotrypsin.Depriving the proteases of their cofactors by poly(acrylic acid) wasreported to induce irreversible structural changes of the enzymeproteins which were accompanied by a loss of enzyme activity. At thesame time, other mucoadhesive polymers (e.g., some cellulose derivativesand chitosan) may not inhibit proteolytic enzymes under certainconditions. In contrast to other enzyme inhibitors contemplated for usewithin the invention (e.g. aprotinin, bestatin), which are relativelysmall molecules, the trans-nasal absorption of inhibitory polymers islikely to be minimal in light of the size of these molecules, andthereby eliminate possible adverse side effects. Thus, mucoadhesivepolymers, particularly of the poly(acrylic acid)-type, may serve both asan absorption-promoting adhesive and enzyme-protective agent to enhancecontrolled delivery of peptide and protein drugs, especially when safetyconcerns are considered.

In addition to protecting against enzymatic degradation, bioadhesivesand other polymeric or non-polymeric absorption-promoting agents for usewithin the invention may directly increase mucosal permeability tobiologically active agents. To facilitate the transport of large andhydrophilic molecules, such as peptides and proteins, across the nasalepithelial barrier, mucoadhesive polymers and other agents have beenpostulated to yield enhanced permeation effects beyond what is accountedfor by prolonged premucosal residence time of the delivery system. Thetime course of drug plasma concentrations reportedly suggested that thebioadhesive microspheres caused an acute, but transient increase ofinsulin permeability across the nasal mucosa. Other mucoadhesivepolymers for use within the invention, for example chitosan, reportedlyenhance the permeability of certain mucosal epithelia even when they areapplied as an aqueous solution or gel. Another mucoadhesive polymerreported to directly affect epithelial permeability is hyaluronic acidand ester derivatives thereof. A particularly useful bioadhesive agentwithin the coordinate administration, and/or combinatorial formulationmethods and compositions of the invention is chitosan, as well as itsanalogs and derivatives. Chitosan is a non-toxic, biocompatible andbiodegradable polymer that is widely used for pharmaceutical and medicalapplications because of its favorable properties of low toxicity andgood biocompatibility. It is a natural polyaminosaccharide prepared fromchitin by N-deacetylation with alkali. As used within the methods andcompositions of the invention, chitosan increases the retention of PTHpeptides, analogs and mimetics, and other biologically active agentsdisclosed herein at a mucosal site of application. This mode ofadministration can also improve patient compliance and acceptance. Asfurther provided herein, the methods and compositions of the inventionwill optionally include a novel chitosan derivative or chemicallymodified form of chitosan. One such novel derivative for use within theinvention is denoted as a β-[1→4]-2-guanidino-2-deoxy-D-glucose polymer(poly-GuD). Chitosan is the N-deacetylated product of chitin, anaturally occurring polymer that has been used extensively to preparemicrospheres for oral and intra-nasal formulations. The chitosan polymerhas also been proposed as a soluble carrier for parenteral drugdelivery. Within one aspect of the invention, o-methylisourea is used toconvert a chitosan amine to its guanidinium moiety. The guanidiniumcompound is prepared, for example, by the reaction between equi-normalsolutions of chitosan and o-methylisourea at pH above 8.0.

The guanidinium product is −[14]-guanidino-2-deoxy-D-glucose polymer. Itis abbreviated as Poly-GuD in this context (Monomer F.W. of Amine inChitosan=161; Monomer F.W. of Guanidinium in Poly-GuD=203).

Additional compounds classified as bioadhesive agents for use within thepresent invention act by mediating specific interactions, typicallyclassified as “receptor-ligand interactions” between complementarystructures of the bioadhesive compound and a component of the mucosalepithelial surface. Many natural examples illustrate this form ofspecific binding bioadhesion, as exemplified by lectin-sugarinteractions. Lectins are (glyco) proteins of non-immune origin whichbind to polysaccharides or glycoconjugates.

Several plant lectins have been investigated as possible pharmaceuticalabsorption-promoting agents. One plant lectin, Phaseolus vulgarishemagglutinin (PHA), exhibits high oral bioavailability of more than 10%after feeding to rats. Tomato (Lycopersicon esculeutum) lectin (TL)appears safe for various modes of administration.

In summary, the foregoing bioadhesive agents are useful in thecombinatorial formulations and coordinate administration methods of theinstant invention, which optionally incorporate an effective amount andform of a bioadhesive agent to prolong persistence or otherwise increasemucosal absorption of one or more PTH peptides, analogs and mimetics,and other biologically active agents. The bioadhesive agents may becoordinately administered as adjunct compounds or as additives withinthe combinatorial formulations of the invention. In certain embodiments,the bioadhesive agent acts as a ‘pharmaceutical glue,’ whereas in otherembodiments adjunct delivery or combinatorial formulation of thebioadhesive agent serves to intensify contact of the biologically activeagent with the nasal mucosa, in some cases by promoting specificreceptor-ligand interactions with epithelial cell “receptors”, and inothers by increasing epithelial permeability to significantly increasethe drug concentration gradient measured at a target site of delivery(e.g., liver, blood plasma, or CNS tissue or fluid). Yet additionalbioadhesive agents for use within the invention act as enzyme (e.g.,protease) inhibitors to enhance the stability of mucosally administeredbiotherapeutic agents delivered coordinately or in a combinatorialformulation with the bioadhesive agent.

The coordinate administration methods and combinatorial formulations ofthe instant invention optionally incorporate effective lipid or fattyacid based carriers, processing agents, or delivery vehicles, to provideimproved formulations for mucosal delivery of PTH peptides, analogs andmimetics, and other biologically active agents. For example, a varietyof formulations and methods are provided for mucosal delivery whichcomprise one or more of these active agents, such as a peptide orprotein, admixed or encapsulated by, or coordinately administered with,a liposome, mixed micellar carrier, or emulsion, to enhance chemical andphysical stability and increase the half life of the biologically activeagents (e.g., by reducing susceptibility to proteolysis, chemicalmodification and/or denaturation) upon mucosal delivery.

Within certain aspects of the invention, specialized delivery systemsfor biologically active agents comprise small lipid vesicles known asliposomes. These are typically made from natural, biodegradable,non-toxic, and non-immunogenic lipid molecules, and can efficientlyentrap or bind drug molecules, including peptides and proteins, into, oronto, their membranes. The attractiveness of liposomes as a peptide andprotein delivery system within the invention is increased by the factthat the encapsulated proteins can remain in their preferred aqueousenvironment within the vesicles, while the liposomal membrane protectsthem against proteolysis and other destabilizing factors. Even thoughnot all liposome preparation methods known are feasible in theencapsulation of peptides and proteins due to their unique physical andchemical properties, several methods allow the encapsulation of thesemacromolecules without substantial deactivation.

A variety of methods are available for preparing liposomes for usewithin the invention, U.S. Pat. Nos. 4,235,871, 4,501,728, and4,837,028, hereby incorporated by reference. For use with liposomedelivery, the biologically active agent is typically entrapped withinthe liposome, or lipid vesicle, or is bound to the outside of thevesicle.

Like liposomes, unsaturated long chain fatty acids, which also haveenhancing activity for mucosal absorption, can form closed vesicles withbilayer-like structures (so called “ufasomes”). These can be formed, forexample, using oleic acid to entrap biologically active peptides andproteins for mucosal, e.g., intranasal, delivery within the invention.

Other delivery systems for use within the invention combine the use ofpolymers and liposomes to ally the advantageous properties of bothvehicles such as encapsulation inside the natural polymer fibrin. Inaddition, release of biotherapeutic compounds from this delivery systemis controllable through the use of covalent crosslinking and theaddition of antifibrinolytic agents to the fibrin polymer.

More simplified delivery systems for use within the invention includethe use of cationic lipids as delivery vehicles or carriers, which canbe effectively employed to provide an electrostatic interaction betweenthe lipid carrier and such charged biologically active agents asproteins and polyanionic nucleic acids. This allows efficient packagingof the drugs into a form suitable for mucosal administration and/orsubsequent delivery to systemic compartments.

Additional delivery vehicles for use within the invention include longand medium chain fatty acids, as well as surfactant mixed micelles withfatty acids. Most naturally occurring lipids in the form of esters haveimportant implications with regard to their own transport across mucosalsurfaces. Free fatty acids and their monoglycerides which have polargroups attached, have been demonstrated in the form of mixed micelles toact on the intestinal barrier as penetration enhancers. This discoveryof barrier modifying function of free fatty acids (carboxylic acids witha chain length varying from 12 to 20 carbon atoms) and their polarderivatives has stimulated extensive research on the application ofthese agents as mucosal absorption enhancers.

For use within the methods of the invention, long chain fatty acids,especially fusogenic lipids (unsaturated fatty acids and monoglyceridessuch as oleic acid, linoleic acid, linoleic acid, monoolein, etc.)provide useful carriers to enhance mucosal delivery of PTH peptide,analogs and mimetics, and other biologically active agents disclosedherein. Medium chain fatty acids (C6 to C12) and monoglycerides havealso been shown to have enhancing activity in intestinal drug absorptionand can be adapted for use within the mocosal delivery formulations andmethods of the invention. In addition, sodium salts of medium and longchain fatty acids are effective delivery vehicles andabsorption-enhancing agents for mucosal delivery of biologically activeagents within the invention. Thus, fatty acids can be employed insoluble forms of sodium salts or by the addition of non-toxicsurfactants, e.g., polyoxyethylated hydrogenated castor oil, sodiumtaurocholate, etc. Other fatty acid and mixed micellar preparations thatare useful within the invention include, but are not limited to, Nacaprylate (C8), Na caprate (C 10), Na laurate (C 12) or Na oleate (C18), optionally combined with bile salts, such as glycocholate andtaurocholate.

Additional methods and compositions provided within the inventioninvolve chemical modification of biologically active peptides andproteins by covalent attachment of polymeric materials, for exampledextrans, polyvinyl pyrrolidones, glycopeptides, polyethylene glycol andpolyamino acids. The resulting conjugated peptides and proteins retaintheir biological activities and solubility for mucosal administration.In alternate embodiments, PTH peptide proteins, analogs and mimetics,and other biologically active peptides and proteins, are conjugated topolyalkylene oxide polymers, particularly polyethylene glycols (PEG).U.S. Pat. No. 4,179,337, hereby incorporated by reference.

Peptides could be linked to PEG directly as described in the art. PEGcan be a molecule having a molecular mass ranging between 300 and60,000. Also included are various PEG molecules, including linear,branched, attached to a peptide at a single moiety or multipleattachment sites. Amine-reactive PEG polymers for use within theinvention include SC-PEG with molecular masses of 2000, 5000, 10000,12000, and 20 000; U-PEG-10000; NHS-PEG-3400-biotin; T-PEG-5000;T-PEG-12000; and TPC-PEG-5000. PEGylation of biologically activepeptides and proteins may be achieved by modification of carboxyl sites(e.g., aspartic acid or glutamic acid groups in addition to the carboxylterminus). The utility of PEG-hydrazide in selective modification ofcarbodiimide-activated protein carboxyl groups under acidic conditionshas been described. Alternatively, bifunctional PEG modification ofbiologically active peptides and proteins can be employed. In someprocedures, charged amino acid residues, including lysine, asparticacid, and glutamic acid, have a marked tendency to be solvent accessibleon protein surfaces.

In addition to PEGylation, biologically active agents such as peptidesand proteins for use within the invention can be modified to enhancecirculating half-life by shielding the active agent via conjugation toother known protecting or stabilizing compounds, for example by thecreation of fusion proteins with an active peptide, protein, analog ormimetic linked to one or more carrier proteins, such as one or moreimmunoglobulin chains.

Mucosal delivery formulations of the present invention comprise PTHpeptides, analogs and mimetics, typically combined together with one ormore pharmaceutically acceptable carriers and, optionally, othertherapeutic ingredients. The carrier(s) must be “pharmaceuticallyacceptable” in the sense of being compatible with the other ingredientsof the formulation and not eliciting an unacceptable deleterious effectin the subject. Such carriers are described herein above or areotherwise well known to those skilled in the art of pharmacology.Desirably, the formulation should not include substances such as enzymesor oxidizing agents with which the biologically active agent to beadministered is known to be incompatible. The formulations may beprepared by any of the methods well known in the art of pharmacy.

Within the compositions and methods of the invention, the PTH peptides,analogs and mimetics, and other biologically active agents disclosedherein may be administered to subjects by a variety of mucosaladministration modes, including by oral, rectal, vaginal, intranasal,intrapulmonary, or transdermal delivery, or by topical delivery to theeyes, ears, skin or other mucosal surfaces. Optionally, PTH peptides,analogs and mimetics, and other biologically active agents disclosedherein can be coordinately or adjunctively administered by non-mucosalroutes, including by intramuscular, subcutaneous, intravenous,intra-atrial, intra-articular, intraperitoneal, or parenteral routes. Inother alternative embodiments, the biologically active agent(s) can beadministered ex vivo by direct exposure to cells, tissues or organsoriginating from a mammalian subject, for example as a component of anex vivo tissue or organ treatment formulation that contains thebiologically active agent in a suitable, liquid or solid carrier.

Compositions according to the present invention are often administeredin an aqueous solution as a nasal or pulmonary spray and may bedispensed in spray form by a variety of methods known to those skilledin the art. Preferred systems for dispensing liquids as a nasal sprayare disclosed in U.S. Pat. No. 4,511,069, hereby incorporated byreference. The formulations may be presented in multi-dose containers,for example in the sealed dispensing system disclosed in U.S. Pat. No.4,511,069. Additional aerosol delivery forms may include, e.g.,compressed air-, jet-, ultrasonic-, and piezoelectric nebulizers, whichdeliver the biologically active agent dissolved or suspended in apharmaceutical solvent, e.g., water, ethanol, or a mixture thereof.

Nasal and pulmonary spray solutions of the present invention typicallycomprise the drug or drug to be delivered, optionally formulated with asurface-active agent, such as a nonionic surfactant (e.g.,polysorbate-80), and one or more buffers. In some embodiments of thepresent invention, the nasal spray solution further comprises apropellant. The pH of the nasal spray solution is optionally betweenabout pH 3.0 and 6.0, preferably 5.0±0.3. Suitable buffers for usewithin these compositions are as described above or as otherwise knownin the art. Other components may be added to enhance or maintainchemical stability, including preservatives, surfactants, dispersants,or gases. Suitable preservatives include, but are not limited to,phenol, methyl paraben, paraben, m-cresol, thiomersal, chlorobutanol,benzylalkonimum chloride, and the like. Suitable surfactants include,but are not limited to, oleic acid, sorbitan trioleate, polysorbates,lecithin, phosphotidyl cholines, and various long chain diglycerides andphospholipids. Suitable dispersants include, but are not limited to,ethylenediaminetetraacetic acid, and the like. Suitable gases include,but are not limited to, nitrogen, helium, chlorofluorocarbons (CFCs),hydrofluorocarbons (HFCs), carbon dioxide, air, and the like.

Within alternate embodiments, mucosal formulations are administered asdry powder formulations comprising the biologically active agent in adry, usually lyophilized, form of an appropriate particle size, orwithin an appropriate particle size range, for intranasal delivery.Minimum particle size appropriate for deposition within the nasal orpulmonary passages is often about 0.5μ mass median equivalentaerodynamic diameter (MMEAD), commonly about 1μ MMEAD, and moretypically about 2μ MMEAD. Maximum particle size appropriate fordeposition within the nasal passages is often about 10μ MMEAD, commonlyabout 8μ MMEAD, and more typically about 4μ MMEAD. Intranasallyrespirable powders within these size ranges can be produced by a varietyof conventional techniques, such as jet milling, spray drying, solventprecipitation, supercritical fluid condensation, and the like. These drypowders of appropriate MMEAD can be administered to a patient via aconventional dry powder inhaler (DPI), which rely on the patient'sbreath, upon pulmonary or nasal inhalation, to disperse the power intoan aerosolized amount. Alternatively, the dry powder may be administeredvia air-assisted devices that use an external power source to dispersethe powder into an aerosolized amount, e.g., a piston pump.

Dry powder devices typically require a powder mass in the range fromabout 1 mg to 20 mg to produce a single aerosolized dose (“puff”). Ifthe required or desired dose of the biologically active agent is lowerthan this amount, the powdered active agent will typically be combinedwith a pharmaceutical dry bulking powder to provide the required totalpowder mass. Preferred dry bulking powders include sucrose, lactose,dextrose, mannitol, glycine, trehalose, human serum albumin (HSA), andstarch. Other suitable dry bulking powders include cellobiose, dextrans,maltotriose, pectin, sodium citrate, sodium ascorbate, and the like.

To formulate compositions for mucosal delivery within the presentinvention, the biologically active agent can be combined with variouspharmaceutically acceptable additives, as well as a base or carrier fordispersion of the active agent(s). Desired additives include, but arenot limited to, pH control agents, such as arginine, sodium hydroxide,glycine, hydrochloric acid, citric acid, etc. In addition, localanesthetics (e.g., benzyl alcohol), isotonizing agents (e.g., sodiumchloride, mannitol, sorbitol), adsorption inhibitors (e.g., Tween 80),solubility enhancing agents (e.g., cyclodextrins and derivativesthereof), stabilizers (e.g., serum albumin), and reducing agents (e.g.,glutathione) can be included. When the composition for mucosal deliveryis a liquid, the tonicity of the formulation, as measured with referenceto the tonicity of 0.9% (w/v) physiological saline solution taken asunity, is typically adjusted to a value at which no substantial,irreversible tissue damage will be induced in the nasal mucosa at thesite of administration. Generally, the tonicity of the solution isadjusted to a value of about ⅓ to 3, more typically ½ to 2, and mostoften ¾ to 1.7.

The biologically active agent may be dispersed in a base or vehicle,which may comprise a hydrophilic compound having a capacity to dispersethe active agent and any desired additives. The base may be selectedfrom a wide range of suitable carriers, including but not limited to,copolymers of polycarboxylic acids or salts thereof, carboxylicanhydrides (e.g., maleic anhydride) with other monomers (e.g., methyl(meth)acrylate, acrylic acid, etc.), hydrophilic vinyl polymers such aspolyvinyl acetate, polyvinyl alcohol, polyvinylpyrrolidone, cellulosederivatives such as hydroxymethylcellulose, hydroxypropylcellulose,etc., and natural polymers such as chitosan, collagen, sodium alginate,gelatin, hyaluronic acid, and nontoxic metal salts thereof Often, abiodegradable polymer is selected as a base or carrier, for example,polylactic acid, poly(lactic acid-glycolic acid) copolymer,polyhydroxybutyric acid, poly(hydroxybutyric acid-glycolic acid)copolymer and mixtures thereof. Alternatively or additionally, syntheticfatty acid esters such as polyglycerin fatty acid esters, sucrose fattyacid esters, etc., can be employed as carriers. Hydrophilic polymers andother carriers can be used alone or in combination, and enhancedstructural integrity can be imparted to the carrier by partialcrystallization, ionic bonding, crosslinking and the like. The carriercan be provided in a variety of forms, including, fluid or viscoussolutions, gels, pastes, powders, microspheres and films for directapplication to the nasal mucosa. The use of a selected carrier in thiscontext may result in promotion of absorption of the biologically activeagent.

The biologically active agent can be combined with the base or carrieraccording to a variety of methods, and release of the active agent maybe by diffusion, disintegration of the carrier, or associatedformulation of water channels. In some circumstances, the active agentis dispersed in microcapsules (microspheres) or nanocapsules(nanospheres) prepared from a suitable polymer, e.g., isobutyl2-cyanoacrylate and dispersed in a biocompatible dispersing mediumapplied to the nasal mucosa, which yields sustained delivery andbiological activity over a protracted time.

To further enhance mucosal delivery of pharmaceutical agents within theinvention, formulations comprising the active agent may also contain ahydrophilic low molecular weight compound as a base or excipient. Suchhydrophilic low molecular weight compounds provide a passage mediumthrough which a water-soluble active agent, such as a physiologicallyactive peptide or protein, may diffuse through the base to the bodysurface where the active agent is absorbed. The hydrophilic lowmolecular weight compound optionally absorbs moisture from the mucosa orthe administration atmosphere and dissolves the water-soluble activepeptide. The molecular weight of the hydrophilic low molecular weightcompound is generally not more than 10000 and preferably not more than3000. Exemplary hydrophilic low molecular weight compound include polyolcompounds, such as oligo-, di- and monosaccarides such as sucrose,mannitol, sorbitol, lactose, L-arabinose, D-erythrose, D-ribose,D-xylose, D-mannose, trehalose, D-galactose, lactulose, cellobiose,gentibiose, glycerin and polyethylene glycol. Other examples ofhydrophilic low molecular weight compounds useful as carriers within theinvention include N-methylpyrrolidone, and alcohols (e.g., oligovinylalcohol, ethanol, ethylene glycol, propylene glycol, etc.). Thesehydrophilic low molecular weight compounds can be used alone or incombination with one another or with other active or inactive componentsof the intranasal formulation.

The compositions of the invention may alternatively contain aspharmaceutically acceptable carriers substances as required toapproximate physiological conditions, such as pH adjusting and bufferingagents, tonicity adjusting agents, wetting agents and the like, forexample, sodium acetate, sodium lactate, sodium chloride, potassiumchloride, calcium chloride, sorbitan monolaurate, triethanolamineoleate, etc. For solid compositions, conventional nontoxicpharmaceutically acceptable carriers can be used which include, forexample, pharmaceutical grades of mannitol, lactose, starch, magnesiumstearate, sodium saccharin, talcum, cellulose, glucose, sucrose,magnesium carbonate, and the like.

Therapeutic compositions for administering the biologically active agentcan also be formulated as a solution, microemulsion, or other orderedstructure suitable for high concentration of active ingredients. Thecarrier can be a solvent or dispersion medium containing, for example,water, ethanol, polyol (for example, glycerol, propylene glycol, andliquid polyethylene glycol, and the like), and suitable mixturesthereof. Proper fluidity for solutions can be maintained, for example,by the use of a coating such as lecithin, by the maintenance of adesired particle size in the case of dispersible formulations, and bythe use of surfactants. In many cases, it will be desirable to includeisotonic agents, for example, sugars, polyalcohols such as mannitol,sorbitol, or sodium chloride in the composition. Prolonged absorption ofthe biologically active agent can be brought about by including in thecomposition an agent which delays absorption, for example, monostearatesalts and gelatin.

In certain embodiments of the invention, the biologically active agentis administered in a time-release formulation, for example in acomposition which includes a slow release polymer. The active agent canbe prepared with carriers that will protect against rapid release, forexample a controlled release vehicle such as a polymer,microencapsulated delivery system or bioadhesive gel. Prolonged deliveryof the active agent, in various compositions of the invention can bebrought about by including in the composition agents that delayabsorption, for example, aluminum monosterate hydrogels and gelatin.When controlled release formulations of the biologically active agent isdesired, controlled release binders suitable for use in accordance withthe invention include any biocompatible controlled-release materialwhich is inert to the active agent and which is capable of incorporatingthe biologically active agent. Numerous such materials are known in theart. Useful controlled-release binders are materials that aremetabolized slowly under physiological conditions following theirintranasal delivery (e.g., at the nasal mucosal surface, or in thepresence of bodily fluids following transmucosal delivery). Appropriatebinders include but are not limited to biocompatible polymers andcopolymers previously used in the art in sustained release formulations.Such biocompatible compounds are non-toxic and inert to surroundingtissues, and do not trigger significant adverse side effects such asnasal irritation, immune response, inflammation, or the like. They aremetabolized into metabolic products that are also biocompatible andeasily eliminated from the body.

Exemplary polymeric materials for use in this context include, but arenot limited to, polymeric matrices derived from copolymeric andhomopolymeric polyesters having hydrolysable ester linkages. A number ofthese are known in the art to be biodegradable and to lead todegradation products having no or low toxicity. Exemplary polymersinclude polyglycolic acids (PGA) and polylactic acids (PLA),poly(DL-lactic acid-co-glycolic acid)(DL PLGA), poly(D-lacticacid-coglycolic acid)(D PLGA) and poly(L-lactic acid-co-glycolic acid)(LPLGA). Other useful biodegradable or bioerodable polymers include butare not limited to such polymers as poly(epsilon-caprolactone),poly(epsilon-aprolactone-CO-lactic acid), poly(-aprolactone-CO-glycolicacid), poly(beta-hydroxy butyric acid), poly(alkyl-2-cyanoacrilate),hydrogels such as poly(hydroxyethyl methacrylate), polyamides,poly(amino acids) (i.e., L-leucine, glutamic acid, L-aspartic acid andthe like), poly (ester urea), poly (2-hydroxyethyl DL-aspartamide),polyacetal polymers, polyorthoesters, polycarbonate, polymaleamides,polysaccharides and copolymers thereof. Many methods for preparing suchformulations are generally known to those skilled in the art. Otheruseful formulations include controlled-release compositions e.g.,microcapsules, U.S. Pat. Nos. 4,652,441 and 4,917,893, lacticacid-glycolic acid copolymers useful in making microcapsules and otherformulations, U.S. Pat. Nos. 4,677,191 and 4,728,721, andsustained-release compositions for water-soluble peptides, U.S. Pat. No.4,675,189, all patents hereby incorporated by reference.

Sterile solutions can be prepared by incorporating the active compoundin the required amount in an appropriate solvent with one or acombination of ingredients enumerated above, as required, followed byfiltered sterilization. Generally, dispersions are prepared byincorporating the active compound into a sterile vehicle that contains abasic dispersion medium and the required other ingredients from thoseenumerated above. In the case of sterile powders, methods of preparationinclude vacuum drying and freeze-drying which yields a powder of theactive ingredient plus any additional desired ingredient from apreviously sterile-filtered solution thereof. The prevention of theaction of microorganisms can be accomplished by various antibacterialand antifungal agents, for example, parabens, chlorobutanol, phenol,sorbic acid, thimerosal, and the like.

Mucosal administration according to the invention allows effectiveself-administration of treatment by patients, provided that sufficientsafeguards are in place to control and monitor dosing and side effects.Mucosal administration also overcomes certain drawbacks of otheradministration forms, such as injections, that are painful and exposethe patient to possible infections and may present drug bioavailabilityproblems. For nasal and pulmonary delivery, systems for controlledaerosol dispensing of therapeutic liquids as a spray are well known. Inone embodiment, metered doses of active agent are delivered by means ofa specially constructed mechanical pump valve, U.S. Pat. No. 4,511,069.

For prophylactic and treatment purposes, the biologically activeagent(s) disclosed herein may be administered to the subjectintranasally once daily. In this context, a therapeutically effectivedosage of the PTH peptide may include repeated doses within a prolongedprophylaxis or treatment regimen that will yield clinically significantresults to alleviate or prevent osteoporosis or osteopenia.Determination of effective dosages in this context is typically based onanimal model studies followed up by human clinical trials and is guidedby determining effective dosages and administration protocols thatsignificantly reduce the occurrence or severity of targeted diseasesymptoms or conditions in the subject. Suitable models in this regardinclude, for example, murine, rat, porcine, feline, non-human primate,and other accepted animal model subjects known in the art.Alternatively, effective dosages can be determined using in vitro models(e.g., immunologic and histopathologic assays). Using such models, onlyordinary calculations and adjustments are typically required todetermine an appropriate concentration and dose to administer atherapeutically effective amount of the biologically active agent(s)(e.g., amounts that are intranasally effective, transdermally effective,intravenously effective, or intramuscularly effective to elicit adesired response). The actual dosage of biologically active agents willof course vary according to factors such as the disease indication andparticular status of the subject (e.g., the subject's age, size,fitness, extent of symptoms, susceptibility factors, etc.), time androute of administration, other drugs or treatments being administeredconcurrently, as well as the specific pharmacology of the biologicallyactive agent(s) for eliciting the desired activity or biologicalresponse in the subject. Dosage regimens may be adjusted to provide anoptimum prophylactic or therapeutic response. A therapeuticallyeffective amount is also one in which any toxic or detrimental sideeffects of the biologically active agent are outweighed in clinicalterms by therapeutically beneficial effects. A non-limiting range for atherapeutically effective amount of a PTH peptide within the methods andformulations of the invention is 0.7 μg/kg to about 25 μg/kg. To treatosteoporosis or osteopenia, an intranasal dose of PTH peptide isadministered at dose high enough to promote the increase in bone massbut low enough so as not to induce any unwanted side-effects such asnausea. A preferred intranasal dose of parathyroid hormone 1-34 is about1 μg-10 μg/kg weight of the patient, most preferably from about 1.5μg/kg to about 3 μg/kg weight of the patient. In a standard dose apatient will receive 50 μg to 1600 μg, more preferably about between 75μg to 800 μg, most preferably 100 μg, 150 μg, 200 μg to about 400 μg.Alternatively, a non-limiting range for a therapeutically effectiveamount of a biologically active agent within the methods andformulations of the invention is between about 0.001 pmol to about 100pmol per kg body weight, between about 0.01 pmol to about 10 pmol per kgbody weight, between about 0.1 pmol to about 5 pmol per kg body weight,or between about 0.5 pmol to about 1.0 pmol per kg body weight. Peradministration, it is desirable to administer at least one microgram ofthe biologically active agent (e.g., one or more PTH peptide proteins,analogs and mimetics, and other biologically active agents), moretypically between about 10 μg and 5.0 mg, and in certain embodimentsbetween about 100 μg and 1.0 or 2.0 mg to an average human subject. Forcertain oral applications, doses as high as 0.5 mg per kg body weightmay be necessary to achieve adequate plasma levels. It is to be furthernoted that for each particular subject, specific dosage regimens shouldbe evaluated and adjusted over time according to the individual need andprofessional judgment of the person administering or supervising theadministration of the permeabilizing peptide(s) and other biologicallyactive agent(s). An intranasal dose of a parathyroid hormone will rangefrom 50 μg to 1600 μg of parathyroid hormone, preferably 75 μg to 800μg, more preferably 100 μg to 400 μg with a most preferred dose beingbetween 100 μg to 200 μg with 150 μg being a dose that is considered tobe highly effective. Repeated intranasal dosing with the formulations ofthe invention, on a schedule ranging from about 0.1 to24 hours betweendoses, preferably between 0.5 and 24.0 hours between doses, willmaintain normalized, sustained therapeutic levels of PTH peptide tomaximize clinical benefits while minimizing the risks of excessiveexposure and side effects. The goal is to mucosally deliver an amount ofthe PTH peptide sufficient to raise the concentration of the PTH peptidein the plasma of an individual to promote increase in bone mass.

Dosage of PTH agonists such as parathyroid hormone may be varied by theattending clinician or patient, if self administering an over thecounter dosage form, to maintain a desired concentration at the targetsite.

In an alternative embodiment, the invention provides compositions andmethods for intranasal delivery of PTH peptide, wherein the PTH peptidecompound(s) is/are repeatedly administered through an intranasaleffective dosage regimen that involves multiple administrations of thePTH peptide to the subject during a daily or weekly schedule to maintaina therapeutically effective elevated and lowered pulsatile level of PTHpeptide during an extended dosing period. The compositions and methodprovide PTH peptide compound(s) that are self-administered by thesubject in a nasal formulation between one and six times daily tomaintain a therapeutically effective elevated and lowered pulsatilelevel of PTH peptide during an 8 hour to 24 hour extended dosing period.

The instant invention also includes kits, packages and multicontainerunits containing the above described pharmaceutical compositions, activeingredients, and/or means for administering the same for use in theprevention and treatment of diseases and other conditions in mammaliansubjects. Briefly, these kits include a container or formulation thatcontains one or more PTH peptide proteins, analogs or mimetics, and/orother biologically active agents in combination with mucosal deliveryenhancing agents disclosed herein formulated in a pharmaceuticalpreparation for mucosal delivery.

The intranasal formulations of the present invention can be administeredusing any spray bottle or syringe. An example of a nasal spray bottle isthe, “Nasal Spray Pump w/Safety Clip,” Pfeiffer SAP #60548, whichdelivers a dose of 0.1 mL per squirt and has a diptube length of 36.05mm. It can be purchased from Pfeiffer of America of Princeton, N.J.Intranasal doses of a PTH peptide such as parathyroid hormone can rangefrom 0.1 μg/kg to about 1500 μg/kg. When administered in as anintranasal spray, it is preferable that the particle size of the sprayis between 10-100 μm (microns) in size, preferably 20-100 μm in size.

We have discovered that the parathyroid hormone peptides can beadministered intranasally using a nasal spray or aerosol. This issurprising because many proteins and peptides have been shown to besheared or denatured due to the mechanical forces generated by theactuator in producing the spray or aerosol. In this area the followingdefinitions are useful.

1. Aerosol—A product that is packaged under pressure and containstherapeutically active ingredients that are released upon activation ofan appropriate valve system.

2. Metered aerosol—A pressurized dosage form comprised of metered dosevalves, which allow for the delivery of a uniform quantity of spray uponeach activation.

3. Powder aerosol—A product that is packaged under pressure and containstherapeutically active ingredients in the form of a powder, which arereleased upon activation of an appropriate valve system.

4. Spray aerosol—An aerosol product that utilizes a compressed gas asthe propellant to provide the force necessary to expel the product as awet spray; it generally applicable to solutions of medicinal agents inaqueous solvents.

5. Spray—A liquid minutely divided as by a jet of air or steam. Nasalspray drug products contain therapeutically active ingredients dissolvedor suspended in solutions or mixtures of excipients in nonpressurizeddispensers.

6. Metered spray—A non-pressurized dosage form consisting of valves thatallow the dispensing of a specified quantity of spray upon eachactivation.

7. Suspension spray—A liquid preparation containing solid particlesdispersed in a liquid vehicle and in the form of course droplets or asfinely divided solids.

The fluid dynamic characterization of the aerosol spray emitted bymetered nasal spray pumps as a drug delivery device (“DDD”). Spraycharacterization is an integral part of the regulatory submissionsnecessary for Food and Drug Administration (“FDA”) approval of researchand development, quality assurance and stability testing procedures fornew and existing nasal spray pumps.

Thorough characterization of the spray's geometry has been found to bethe best indicator of the overall performance of nasal spray pumps. Inparticular, measurements of the spray's divergence angle (plumegeometry) as it exits the device; the spray's cross-sectionalellipticity, uniformity and particle/droplet distribution (spraypattern); and the time evolution of the developing spray have been foundto be the most representative performance quantities in thecharacterization of a nasal spray pump. During quality assurance andstability testing, plume geometry and spray pattern measurements are keyidentifiers for verifying consistency and conformity with the approveddata criteria for the nasal spray pumps.

DEFINITIONS

Plume Height—the measurement from the actuator tip to the point at whichthe plume angle becomes non-linear because of the breakdown of linearflow. Based on a visual examination of digital images, and to establisha measurement point for width that is consistent with the farthestmeasurement point of spray pattern, a height of 30 mm is defined forthis study

Major Axis—the largest chord that can be drawn within the fitted spraypattern that crosses the COMw in base units (mm)

Minor Axis—the smallest chord that can be drawn within the fitted spraypattern that crosses the COMw in base units (mm)

Ellipticity Ratio—the ratio of the major axis to the minor axis

D₁₀—the diameter of droplet for which 10% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm)

D₅₀—the diameter of droplet for which 50% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm), also known asthe mass median diameter

D₉₀—the diameter of droplet for which 90% of the total liquid volume ofsample consists of droplets of a smaller diameter (μm)

Span—measurement of the width of the distribution, The smaller thevalue, the narrower the distribution. Span is calculated as

$\frac{\left( {D_{90} - D_{10}} \right)}{D_{50}}.$

% RSD—percent relative standard deviation, the standard deviationdivided by the mean of the series and multiplied by 100, also known as %CV.

A nasal spray device can be selected according to what is customary inthe industry or acceptable by the regulatory health authorities. Oneexample of a suitable device is described in described in U.S.application Ser. No. 10/869,649 (S. Quay and G. Brandt: Compositions andMethods for Enhanced Mucosal Delivery of Y2 Receptor-binding Peptidesand Methods for Treating and Preventing Obesity, filed Jun. 16, 2004).

To treat osteoporosis or osteopenia, an intranasal dose of a PTH peptideparathyroid hormone is administered at dose high enough to promote anincrease in bone mass but low enough so as not to induce any unwantedside-effects such as nausea. A preferred intranasal dose of a PTHpeptide such as parathyroid hormone(1-34) is about 3 μg-10 μg/kg weightof the patient, most preferably about 6 μg/kg weight of the patient. Ina standard dose a patient will receive 50 μg to 800 μg, more preferablyabout between 100 μg to 400 μg, most preferably 150 μg to about 200 μg.The a PTH peptide such as parathyroid hormone (1-34) is preferablyadministered once a day.

The following examples are provided by way of illustration, notlimitation.

EXAMPLE 1

An exemplary formulation for enhanced nasal mucosal delivery of PTHfollowing the teachings of the instant specification can be prepared andevaluated as shown in Table 1.

EXAMPLE 2 Nasal Mucosal Delivery—Permeation Kinetics and Cytotoxicity

The following methods are generally useful for evaluating nasal mucosaldelivery parameters, kinetics and side effects for PTH within theformulations and method of the invention, as well as for determining theefficacy and characteristics of the various intranasaldelivery-enhancing agents disclosed herein for combinatorial formulationor coordinate administration with PTH.

Permeation kinetics and cytotoxicity are also useful for determining theefficacy and characteristics of the various mucosal delivery-enhancingagents disclosed herein for combinatorial formulation or coordinateadministration with mucosal delivery-enhancing agents. In one exemplaryprotocol, permeation kinetics and lack of unacceptable cytotoxicity aredemonstrated for an intranasal delivery-enhancing agent as disclosedabove in combination with a biologically active therapeutic agent,exemplified by PTH.

The EPIAIRWAY® system was developed by MatTek Corp (Ashland, Mass.) as amodel of the pseudostratified epithelium lining the respiratory tract.The epithelial cells are grown on porous membrane-bottomed cell cultureinserts at an air-liquid interface, which results in differentiation ofthe cells to a highly polarized morphology. The apical surface isciliated with a microvillous ultrastructure and the epithelium producesmucus (the presence of mucin has been confirmed by immunoblotting). Theinserts have a diameter of 0.875 cm, providing a surface area of 0.6cm². The cells are plated onto the inserts at the factory approximatelythree weeks before shipping.

On arrival, the units are placed onto sterile supports in 6-wellmicroplates. Each well receives 5 mL of proprietary culture medium. ThisDMEM-based medium is serum free but is supplemented with epidermalgrowth factor and other factors. The medium is always tested forendogenous levels of any cytokine or growth factor, which is beingconsidered for intranasal delivery, but has been free of all cytokinesand factors studied to date except insulin. The 5 mL volume is justsufficient to provide contact to the bottoms of the units on theirstands, but the apical surface of the epithelium is allowed to remain indirect contact with air. Sterile tweezers are used in this step and inall subsequent steps involving transfer of units to liquid-containingwells to ensure that no air is trapped between the bottoms of the unitsand the medium.

The units in their plates are maintained at 37° C. in an incubator in anatmosphere of 5% CO₂ in air for 24 hours. At the end of this time themedium is replaced with fresh medium and the units are returned to theincubator for another 24 hours.

A “kit” of 24 EPIAIRWAY® units can routinely be employed for evaluatingfive different formulations, each of which is applied to quadruplicatewells. Each well is employed for determination of permeation kinetics (4time points), transepithelial resistance, mitochondrial reductaseactivity as measured by MTT reduction, and cytolysis as measured byrelease of LDH. An additional set of wells is employed as controls,which are sham treated during determination of permeation kinetics, butare otherwise handled identically to the test sample-containing unitsfor determinations of transepithelial resistance and viability. Thedeterminations on the controls are routinely also made on quadruplicateunits, but occasionally we have employed triplicate units for thecontrols and have dedicated the remaining four units in the kit tomeasurements of transepithelial resistance and viability on untreatedunits or we have frozen and thawed the units for determinations of totalLDH levels to serve as a reference for 100% cytolysis.

In all experiments, the nasal mucosal delivery formulation to be studiedis applied to the apical surface of each unit in a volume of 100 μL,which is sufficient to cover the entire apical surface. An appropriatevolume of the test formulation at the concentration applied to theapical surface (no more than 100 μL is generally needed) is set asidefor subsequent determination of concentration of the active material byELISA or other designated assay.

The units are placed in 6 well plates without stands for the experiment:each well contains 0.9 mL of medium which is sufficient to contact theporous membrane bottom of the unit but does not generate any significantupward hydrostatic pressure on the unit.

To minimize potential sources of error and avoid any formation ofconcentration gradients, the units are transferred from one 0.9mL-containing well to another at each time point in the study. Thesetransfers are made at the following time points, based on a zero time atwhich the 100 μL volume of test material was applied to the apicalsurface: 15 minutes, 30 minutes, 60 minutes, and 120 minutes.

In between time points the units in their plates are kept in the 37° C.incubator. Plates containing 0.9 mL medium per well are also maintainedin the incubator so that minimal change in temperature occurs during thebrief periods when the plates are removed and the units are transferredfrom one well to another using sterile forceps.

At the completion of each time point, the medium is removed from thewell from which each unit was transferred, and aliquotted into two tubes(one tube receives 700 μL and the other 200 μL) for determination of theconcentration of permeated test material and, in the event that the testmaterial is cytotoxic, for release of the cytosolic enzyme, lacticdehydrogenase, from the epithelium. These samples are kept in therefrigerator if the assays are to be conducted within 24 hours, or thesamples are subaliquotted and kept frozen at −80° C. until thawed oncefor assays. Repeated freeze-thaw cycles are to be avoided.

In order to minimize errors, all tubes, plates, and wells are prelabeledbefore initiating an experiment.

At the end of the 120 minute time point, the units are transferred fromthe last of the 0.9 mL containing wells to 24-well microplates,containing 0.3 mL medium per well. This volume is again sufficient tocontact the bottoms of the units, but not to exert upward hydrostaticpressure on the units. The units are returned to the incubator prior tomeasurement of transepithelial resistance.

Respiratory airway epithelial cells form tight junctions in vivo as wellas in vitro, restricting the flow of solutes across the tissue. Thesejunctions confer a transepithelial resistance of several hundredohms×cm² in excised airway tissues; in the MatTek EpiAirway units, thetransepithelial resistance (TER) is claimed by the manufacturer to beroutinely around 1000 ohms×cm². We have found that the TER of controlEPIAIRWAY® units which have been sham-exposed during the sequence ofsteps in the permeation study is somewhat lower (700-800 ohms×cm²), but,since permeation of small molecules is proportional to the inverse ofthe TER, this value is still sufficiently high to provide a majorbarrier to permeation. The porous membrane-bottomed units without cells,conversely, provide only minimal transmembrane resistance (5-20ohms×cm²).

Accurate determinations of TER require that the electrodes of theohmmeter be positioned over a significant surface area above and belowthe membrane, and that the distance of the electrodes from the membranebe reproducibly controlled. The method for TER determination recommendedby MatTek and employed for all experiments here employs an “EVOM™”epithelial voltohmmeter and an “ENDOHM™” tissue resistance measurementchamber from World Precision Instruments, Inc., Sarasota, Fla.

The chamber is initially filled with Dulbecco's phosphate bufferedsaline (PBS) for at least 20 minutes prior to TER determinations inorder to equilibrate the electrodes.

Determinations of TER are made with 1.5 mL of PBS in the chamber and 350μL of PBS in the membrane-bottomed unit being measured. The topelectrode is adjusted to a position just above the membrane of a unitcontaining no cells (but containing 350 μL of PBS) and then fixed toensure reproducible positioning. The resistance of a cell-free unit istypically 5-20 ohms×cm² (“background resistance”).

Once the chamber is prepared and the background resistance is recorded,units in a 24-well plate which had just been employed in permeationdeterminations are removed from the incubator and individually placed inthe chamber for TER determinations.

Each unit is first transferred to a petri dish containing PBS to ensurethat the membrane bottom is moistened. An aliquot of 350 μL PBS is addedto the unit and then carefully aspirated into a labeled tube to rinsethe apical surface. A second wash of 350 μL PBS is then applied to theunit and aspirated into the same collection tube.

The unit is blotted free of excess PBS on its exterior surface onlybefore being placed into the chamber (containing a fresh 1.5 mL aliquotof PBS). An aliquot of 350 μL PBS is added to the unit before the topelectrode is placed on the chamber and the TER is read on the EVOMmeter.

After the TER of the unit is read in the ENDOHM chamber, the unit isremoved, the PBS is aspirated and saved, and the unit is returned withan air interface on the apical surface to a 24-well plate containing 0.3mL medium per well.

The units are read in the following sequence: all sham-treated controls,followed by all formulation-treated samples, followed by a second TERreading of each of the sham-treated controls. After all the TERdeterminations are complete, the units in the 24-well microplate arereturned to the incubator for determination of viability by MTTreduction.

MTT is a cell-permeable tetrazolium salt which is reduced bymitochondrial dehydrogenase activity to an insoluble colored formazan byviable cells with intact mitochondrial function or by nonmitochondrialNAD(P)H dehydrogenase activity from cells capable of generating arespiratory burst. Formation of formazan is a good indicator ofviability of epithelial cells since these cells do not generate asignificant respiratory burst. We have employed a MTT reagent kitprepared by MatTek Corp for their units in order to assess viability.

The MTT reagent is supplied as a concentrate and is diluted into aproprietary DMEM-based diluent on the day viability is to be assayed(typically the afternoon of the day in which permeation kinetics and TERwere determined in the morning). Insoluble reagent is removed by a briefcentrifugation before use. The final MTT concentration is 1 mg/mL.

The final MTT solution is added to wells of a 24-well microplate at avolume of 300 μL per well. As has been noted above, this volume issufficient to contact the membranes of the EPIAIRWAY® units but imposesno significant positive hydrostatic pressure on the cells.

The units are removed from the 24-well plate in which they were placedafter TER measurements, and after removing any excess liquid from theexterior surface of the units, they are transferred to the platecontaining MTT reagent. The units in the plate are then placed in anincubator at 37° C. in an atmosphere of 5% CO₂ in air for 3 hours.

At the end of the 3-hour incubation, the units containing viable cellswill have turned visibly purple. The insoluble formazan must beextracted from the cells in their units to quantitate the extent of MTTreduction. Extraction of the formazan is accomplished by transferringthe units to a 24-well microplate containing 2 mL extractant solutionper well, after removing excess liquid from the exterior surface of theunits as before. This volume is sufficient to completely cover both themembrane and the apical surface of the units. Extraction is allowed toproceed overnight at room temperature in a light-tight chamber. MTTextractants traditionally contain high concentrations of detergent, anddestroy the cells.

At the end of the extraction, the fluid from within each unit and thefluid in its surrounding well are combined and transferred to a tube forsubsequent aliquotting into a 96-well microplate (200 μL aliquots areoptimal) and determination of absorbance at 570 nm on a VMax multiwellmicroplate spectrophotometer. To ensure that turbidity from debriscoming from the extracted units does not contribute to the absorbance,the absorbance at 650 nm is also determined for each well in the VMaxand is automatically subtracted from the absorbance at 570 nm. The“blank” for the determination of formazan absorbance is a 200 μL aliquotof extractant to which no unit had been exposed. This absorbance valueis assumed to constitute zero viability.

Two units from each kit of 24 EPIAIRWAY® units are left untreated duringdetermination of permeation kinetics and TER. These units are employedas the positive control for 100% cell viability. In all the studies wehave conducted, there has been no statistically significant differencein the viability of the cells in these untreated units vs cells incontrol units which had been sham treated for permeation kinetics and onwhich TER determinations had been performed. The absorbance of all unitstreated with test formulations is assumed to be linearly proportional tothe percent viability of the cells in the units at the time of theincubation with MTT. It should be noted that this assay is carried outtypically no sooner than four hours after introduction of the testmaterial to the apical surface, and subsequent to rinsing of the apicalsurface of the units during TER determination.

While measurement of mitochondrial reductase activity by MTT reductionis a sensitive probe of cell viability, the assay necessarily destroysthe cells and therefore can be carried out only at the end of eachstudy. When cells undergo necrotic lysis, their cytotosolic contents arespilled into the surrounding medium, and cytosolic enzymes such aslactic dehydrogenase (LDH) can be detected in this medium. An assay forLDH in the medium can be performed on samples of medium removed at eachtime point of the two-hour determination of permeation kinetics. Thus,cytotoxic effects of formulations which do not develop until significanttime has passed can be detected as well as effects of formulations whichinduce cytolysis with the first few minutes of exposure to airwayepithelium.

The recommended LDH assay for evaluating cytolysis of the EPIAIRWAY®units is based on conversion of lactate to pyruvate with generation ofNADH from NAD. The NADH is then reoxidized along with simultaneousreduction of the tetrazolium salt INT, catalyzed by a crude “diaphorase”preparation. The formazan formed from reduction of INT is soluble, sothat the entire assay for LDH activity can be carried out in ahomogenous aqueous medium containing lactate, NAD, diaphorase, and INT.

The assay for LDH activity is carried out on 50 μL aliquots from samplesof “supernatant” medium surrounding an EPIAIRWAY® unit and collected ateach time point. These samples were either stored for no longer than 24h in the refrigerator or were thawed after being frozen within a fewhours after collection. Each EPIAIRWAY® unit generates samples ofsupernatant medium collected at 15 min, 30 min, 1 h, and 2 h afterapplication of the test material. The aliquots are all transferred to a96 well microplate.

A 50 μL aliquot of medium which had not been exposed to a unit serves asa “blank” or negative control of 0% cytotoxicity. We have found that theapparent level of “endogenous” LDH present after reaction of the assayreagent mixture with the unexposed medium is the same withinexperimental error as the apparent level of LDH released by all thesham-treated control units over the entire time course of 2 hoursrequired to conduct a permeation kinetics study. Thus, withinexperimental error, these sham-treated units show no cytolysis of theepithelial cells over the time course of the permeation kineticsmeasurements.

To prepare a sample of supernatant medium reflecting the level of LDHreleased after 100% of the cells in a unit have lysed, a unit which hadnot been subjected to any prior manipulations is added to a well of a6-well microplate containing 0.9 mL of medium as in the protocol fordetermination of permeation kinetics, the plate containing the unit isfrozen at −80° C., and the contents of the well are then allowed tothaw. This freeze-thaw cycle effectively lyses the cells and releasestheir cytosolic contents, including LDH, into the supernatant medium. A50 μL aliquot of the medium from the frozen and thawed cells is added tothe 96-well plate as a positive control reflecting 100% cytotoxicity.

To each well containing an aliquot of supernatant medium, a 50 μLaliquot of the LDH assay reagent is added. The plate is then incubatedfor 30 minutes in the dark.

The reactions are terminated by addition of a “stop” solution of 1 Macetic acid, and within one hour of addition of the stop solution, theabsorbance of the plate is determined at 490 nm.

Computation of percent cytolysis is based on the assumption of a linearrelationship between absorbance and cytolysis, with the absorbanceobtained from the medium alone serving as a reference for 0% cytolysisand the absorbance obtained from the medium surrounding a frozen andthawed unit serving as a reference for 100% cytolysis.

The procedures for determining the concentrations of biologically activeagents as test materials for evaluating enhanced permeation of activeagents in conjunction with coordinate administration of mucosaldelivery-enhancing agents or combinatorial formulation of the inventionare generally as described above and in accordance with known methodsand specific manufacturer instructions of ELISA kits employed for eachparticular assay. Permeation kinetics of the biologically active agentis generally determined by taking measurements at multiple time points(for example 15 min., 30 min., 60 min. and 120 min) after thebiologically active agent is contacted with the apical epithelial cellsurface (which may be simultaneous with, or subsequent to, exposure ofthe apical cell surface to the mucosal delivery-enhancing agent(s)).

The procedures for determining the concentrations of PTH peptide inblood serum, central nervous system (CNS) tissues or fluids, cerebralspinal fluid (CSF), or other tissues or fluids of a mammalian subjectmay be determined by immunologic assay for PTH. The procedures fordetermining the concentrations of PTH as test materials for evaluatingenhanced permeation of active agents in conjunction with coordinateadministration of mucosal delivery-enhancing agents or combinatorialformulation of the invention are generally as described above and inaccordance with known methods and specific manufacturer instructions forradioimmunoassay (RIA), enzyme immunoassay (EIA), and antibody reagentsfor immunohistochemistry or immunofluorescence for PTH peptide. BachemAG (King of Prussia, Pa.).

EPIAIRWAY® tissue membranes are cultured in phenol red andhydrocortisone free medium (MatTek Corp., Ashland, Mass.). The tissuemembranes are cultured at 37° C. for 48 hours to allow the tissues toequilibrate. Each tissue membrane is placed in an individual well of a6-well plate containing 0.9 mL of serum free medium. 100 μL of theformulation (test sample or control) is applied to the apical surface ofthe membrane. Triplicate or quadruplicate samples of each test sample(mucosal delivery-enhancing agent in combination with a biologicallyactive agent, PTH) and control (biologically active agent, PTH, alone)are evaluated in each assay. At each time point (15, 30, 60 and 120minutes) the tissue membranes are moved to new wells containing freshmedium. The underlying 0.9 mL medium samples is harvested at each timepoint and stored at 4° C. for use in ELISA and lactate dehydrogenase(LDH) assays.

The ELISA kits are typically two-step sandwich ELISAs: theimmunoreactive form of the agent being studied is first “captured” by anantibody immobilized on a 96-well microplate and after washing unboundmaterial out of the wells, a “detection” antibody is allowed to reactwith the bound immunoreactive agent. This detection antibody istypically conjugated to an enzyme (most often horseradish peroxidase)and the amount of enzyme bound to the plate in immune complexes is thenmeasured by assaying its activity with a chromogenic reagent. Inaddition to samples of supernatant medium collected at each of the timepoints in the permeation kinetics studies, appropriately diluted samplesof the formulation (i.e., containing the subject biologically activetest agent) that was applied to the apical surface of the units at thestart of the kinetics study are also assayed in the ELISA plate, alongwith a set of manufacturer-provided standards. Each supernatant mediumsample is generally assayed in duplicate wells by ELISA (it will berecalled that quadruplicate units are employed for each formulation in apermeation kinetics determination, generating a total of sixteen samplesof supernatant medium collected over all four time points).

It is not uncommon for the apparent concentrations of active test agentin samples of supernatant medium or in diluted samples of materialapplied to the apical surface of the units to lie outside the range ofconcentrations of the standards after completion of an ELISA. Noconcentrations of material present in experimental samples aredetermined by extrapolation beyond the concentrations of the standards;rather, samples are rediluted appropriately to generate concentrationsof the test material which can be more accurately determined byinterpolation between the standards in a repeat ELISA.

The ELISA for a biologically active test agent, for example, PTH, isunique in its design and recommended protocol. Unlike most kits, theELISA employs two monoclonal antibodies, one for capture and another,directed towards a nonoverlapping determinant for the biologicallyactive test agent, e.g., PTH, as the detection antibody (this antibodyis conjugated to horseradish peroxidase). As long as concentrations ofPTH that lie below the upper limit of the assay are present inexperimental samples, the assay protocol can be employed as per themanufacturer's instructions, which allow for incubation of the sampleson the ELISA plate with both antibodies present simultaneously. When thePTH levels in a sample are significantly higher than this upper limit,the levels of immunoreactive PTH may exceed the amounts of theantibodies in the incubation mixture, and some PTH which has nodetection antibody bound will be captured on the plate, while some PTHwhich has detection antibody bound may not be captured. This leads toserious underestimation of the PTH levels in the sample (it will appearthat the PTH levels in such a sample lie significantly below the upperlimit of the assay). To eliminate this possibility, the assay protocolhas been modified:

The diluted samples are first incubated on the ELISA plate containingthe immobilized capture antibody for one hour in the absence of anydetection antibody. After the one hour incubation, the wells are washedfree of unbound material.

The detection antibody is incubated with the plate for one hour topermit formation of immune complexes with all captured antigen. Theconcentration of detection antibody is sufficient to react with themaximum level of PTH which has been bound by the capture antibody. Theplate is then washed again to remove any unbound detection antibody.

The peroxidase substrate is added to the plate and incubated for fifteenminutes to allow color development to take place.

The “stop” solution is added to the plate, and the absorbance is read at450 nm as well as 490 nm in the VMax microplate spectrophotometer. Theabsorbance of the colored product at 490 nm is much lower than that at450 nm, but the absorbance at each wavelength is still proportional toconcentration of product. The two readings ensure that the absorbance islinearly related to the amount of bound PTH over the working range ofthe VMax instrument (we routinely restrict the range from 0 to 2.5 OD,although the instrument is reported to be accurate over a range from 0to 3.0 OD). The amount of PTH in the samples is determined byinterpolation between the OD values obtained for the different standardsincluded in the ELISA. Samples with OD readings outside the rangeobtained for the standards are rediluted and run in a repeat ELISA.

Measurement of Transepithelial Resistance by TER Assay

After the final assay time points, membranes are placed in individualwells of a 24-well culture plate in 0.3 mL of clean medium and the transepithelial electrical resistance (TER) is measured using the EVOMEpithelial Voltohmmeter and an Endohm chamber (World PrecisionInstruments, Sarasota, Fla.). The top electrode is adjusted to be closeto, but not in contact with, the top surface of the membrane. Tissuesare removed, one at a time, from their respective wells and basalsurfaces are rinsed by dipping in clean PBS. Apical surfaces were gentlyrinsed twice with PBS. The tissue unit is placed in the Endohm chamber,250 μL of PBS added to the insert, the top electrode replaced and theresistance measured and recorded. Following measurement, the PBS isdecanted and the tissue insert is returned to the culture plate. All TERvalues are reported as a function of the surface area of the tissue.

The final numbers are calculated as:

TER of cell membrane=(Resistance (R) of Insert with membrane−R of blankInsert)×Area of membrane (0.6 cm²).

EXAMPLE 3 Preparation of a Parathyroid Hormone Formulation Free of aStabilizer that is a Protein

A parathyroid hormone formulation suitable for intranasal administrationof parathyroid hormone, which was substantially free of a stabilizerthat is a protein was prepared having the formulation listed below.

About ¾ of the water was added to a beaker and stirred with a stir baron a stir plate and the sodium citrate was added until it was completelydissolved. The EDTA was then added and stirred until it was completelydissolved. The citric acid was then added and stirred until it wascompletely dissolved. The methyl-β-cyclodextrin was added and stirreduntil it was completely dissolved. The DDPC was then added and stirreduntil it was completely dissolved. The lactose was then added andstirred until it was completely dissolved. The sorbitol was then addedand stirred until it was completely dissolved. The chlorobutanol wasthen added and stirred until it was completely dissolved. Theparathyroid hormone 1-34 was added and stirred gently until itdissolved. The pH was adjuste to 5.0±0.25 by addition of HCl or NaOH.Water was added to final volume.

EXAMPLE 4 PTH and Enhancer Effects on Human Chondrocytes

The effect of intranasal PTH₁₋₃₄ formulation on human chondrocytes wasmeasured in vitro, specifically measuring the effect of permeationenhancers on chondrocyte proliferation and production of collagen incomparison to calcitonin and IGF-1.

The cell lines used were derived from human articular cartilage.Articular chondrocytes are phenotypically very similar to nasalchondrocytes (Shikani et al., 2004), and thus provided a good model forthe current study. The cells were provided in two different forms, thefirst as a monolayer (proliferation model) and the second as cellsencapsulated in alginate beads (redifferention model).

The cell monolayer model was employed to examine cell proliferation. Theobjective was to examine cell proliferation in the presence of PTH₁₋₃₄in a simple formulation (citrate buffer) or a formulation containingformulation enhancers, and then compare these data to cell proliferationfor a positive control (media containing antibiotics, insulin, TGF-betaand IGF-1) and negative control (media devoid of any cell growthcomponents). A placebo solution used to make peptide-containingformulations is described in Table 3. Peptide was added to this solutionto achieve the desired concentration. PTH₁₋₃₄ and salmon calcitonin usedwere from Nastech. IGF-1 was purchased from Sigma.

Chondrocyte monolayers (Cell Applications, Inc., San Diego, Calif.)derived from normal human cartilage were adhered on to a 24-well plateand shipped following the first doubling. Approximately 16000 cells perwell were expected at the time the cells were received. Two plates weretreated identically with PTH₁₋₃₄ and appropriate control samples.Controls included cells treated with chondrocyte growth media forpositive control (Cell Applications) and cells treated with basal mediafor negative control (Cell Applications).

Once treated, one of the two plates was analyzed for cell viability (t=0sample). The second plate was placed at 37° C., 5% CO₂ incubator for 4days, after which the plates were analyzed using the MTT assay.

For MTT analysis (Cell Applications), a volume of 100 μL MTT concentratesolution was added to each well containing 1000 μL sample. Plates weresealed and placed at 37° C. for 4 hours. Each plate was removed from 37°C. after 4 h incubation and placed on a bench top. Supernatant from eachwell was carefully removed and discarded. Visible purple crystals wereseen attached to the bottom of the plated in each well. A volume of 500μL of extraction solution was then added into each well. Plates weresealed with parafilm immediately after adding extraction solution, andthen gently rocked and/or swirled to solubilize the purple crystals.Absorbance was read at 570 nm.

Approximately 3 million human chondrocyte cells (Cell Applications)encapsulated in alginate beads were received in 25 mL total volume ofre-differentiated medium. Human chondrocytes in alginate beads producetheir phenotypic markers such as aggrecan and Type II collagen (Benya etal., 1982; Guo et al., 1982; Kato et al., 1984) unlike in monolayerculture where chondrocytes lose their phenotypic characteristics andde-differentiate to fibroblast-like cells (Kuettmer et al., 1982;Jennings et al., 1983; Kato et al., 1984). Alginate based cell systemwas pursued to assess Type II collagen expression as result of PTH₁₋₃₄dosing and therefore indication of any cartilage growth.

The cell-containing alginate bead suspension was transferred from cellculture flask to 50 mL conical tubes. A volume of 500 μL (˜62,500 cellsper well) suspension was dispensed per well in 24-well plates. Sampleswere added to each well and final volume was brought to one milliliterwith appropriate growth media. Controls included cells treated withre-differentiated media as positive control (Cell Applications), cellstreated with chondrocyte growth media (Cell Applications) and basalmedia (Cell Applications) as negative controls.

Plates were swirled gently to allow homogenous mixture of each sample.Plates were placed in 37° C., 5% CO₂ incubator. Human chondrocytes weregrown for up to 12 days on longer. Appropriate growth media werereplaced every second or third day. Alginate beads were prepared forType II collagen extraction on the last day of the experiment andextracted collagen was quantitated using Native Type II CollagenDetection Kit (Chondrex Inc., Redmond Wash.). A capture ELISA kit(Chondrex) was used to measure native type II collagen.Glycosaminoglycans were measured by a kit (Accurate Chemical &Scientific Corp.).

The MTT assay was employed to test for cell proliferation. The MTT assaymeasures cell viability, and an increase or decrease in the MTT assayvalues reflect an increase or decrease in the population of viablecells. To test for stimulation of the growth of chondrocytes, varioustest solutions containing PTH₁₋₃₄ were applied to the apical side of thechondrocyte monolayers, and the MTT assay was conducted at the beginningand then after 4 days incubation at 37 ° C./5% CO₂. The results showedthat PTH₁₋₃₄ did not stimulate the growth of chondrocytes whetherformulated as a simple solution or in the presence of permeationenhancers.

A cartilage growth model was examined, in which cells were provided in aform where they were encapsulated in alginate beads. In this form, humanchondrocytes exhibit their phenotypic markers such as aggrecan and TypeII collagen (Benya et al., 1982; Guo et al., 1982; Kato et al., 1984)unlike in monolayer culture where chondrocytes lose their phenotypiccharacteristics and de-differentiate to fibroblast-like cells (Kuettmeret al., 1982; Jennings et al., 1983; Kato et al., 1984). Thisalginate-based cell system was used to assess effect of PTH₁₋₃₄ dosingon Type II collagen expression as an indication of cartilage growth.

PTH₁₋₃₄ was tested for its ability to stimulate chondrocytes to producecartilage. Cell-containing alginate beads were incubated in the presenceof various test solutions for 12 days at 37° C., 5% CO₂. After theincubation, the alginate beads were processed using an extraction methodin order to quantitate the production of Type II collagen (a majorcomponent of extracellular matrix of nasal cartilage (Shikani et al.,2004)).

Production of type II collagen was measured in positive(re-differentiation media) and negative (growth media) controls as wellas the effect of exposing the cells to either 20 or 200 μg PTH₁₋₃₄. Lowlevels of type II collagen were produced in the presence of there-differentiation media but not in the growth media. Application of 20mg of PTH₁₋₃₄ did not cause production of type II collagen from thechondrocytes, either in citrate buffer or with permeation enhancers.When 200 mg of PTH₁₋₃₄ was applied to the cell culture in a simpleformulation, the production of type II collagen was increased.Surprisingly, when the same amount of peptide was given in theformulation containing permeation enhancers, type II collagen was notproduced by the cells. As a control, the cell system was validated intests showing that production of type II collagen was increased byculturing with 5 mg IGF-I.

These results are consistent with a role of permeation enhancers such asmethyl-b-cyclodextrin and/or DDPC in blocking the local activity of thePTH₁₋₃₄.

EXAMPLE 5 PK Study of PTH₁₋₃₄ in Rabbits

The objective of this study was to determine the plasma pharmacokineticsand relative intranasal and subcutaneous bioavailability of PTH₁₋₃₄following intranasal or subcutaneous dose administration to rabbits. Inaddition, the absorption profile of subcutaneous recombinant andsynthetic PTH₁₋₃₄was evaluated.

This was a randomized, single treatment parallel study in four groups offour animals per group. Following dose administration, serial bloodsamples were obtained from each animal by direct venipuncture of amarginal ear vein. Serial blood samples (about 2 mL each) were collectedby direct venipuncture from a marginal ear vein into blood collectiontubes containing EDTA as the anticoagulant. After collection of theblood, the tubes were gently rocked several times immediately forani-coagulation. Aprotinin at 100 μL was added to the collection tubes.Blood samples were collected at 0, 5, 10, 20, 30, 45, 60, 120 and 240minutes post-dosing. Clinical observations were observed at least oncedaily and at all times of blood sampling.

Doses were based on the most recently recorded body weight. Forintranasal and subcutaneous administration animals were dosed at thevolume of 50 μl/kg and 5 μl/kg, respectively. The dose multiples werebased on a nasal surface measurements of the rabbit and human. The nasalsurface dose multiples of the rabbit compared to the human isapproximately 2 fold in this study based on a unilateral nasal surfacearea of 30 cm² and 80 cm² for rabbit and humans, respectively. Thedosing groups are presented in Table 4.

The nasal and subcutaneous formulations for Group 1, 2, 3 and 4 wereaccording to the methods described above. An enzyme immunoassay wasdeveloped to measure the concentration of Human PTH₁₋₃₄ in rabbit serum.Samples were collected with protease inhibitor (aprotinin) and frozen.Samples, Standards, and Quality Control samples are assayed using amodified Human Bioactive PTH₁₋₃₄ ELISA kit. Standards, Samples, andQuality Control samples are added to streptavidin coated strip wells induplicate. These samples are then incubated with a mixture ofbiotinylated human PTH₁₋₃₄ antibody and HRP-conjugated human PTH₁₋₃₄antibody. The plate is washed with the kit Wash solution and TMBsubstrate solution is added to each well. Color is allowed to developfor 10 minutes before solution is added to each well. OD is measured onan absorbance plate reader. Concentration is calculated by interpolationof a standard curve and assay performance is controlled with QualityControl samples. Pharmacokinetic calculations were performed usingWinNonLin software (Pharsight Corporation, Version 4.0, Mountain View,Calif.) using a non-compartmental model.

Group 1 and 2 were dosed at 50 μg/kg by the intranasal route usingsynthetic PTH from Bachem as the active ingredient. Group 1 was themarketed FORTEO® formulation and Group 2 was Nastech's formulation.Group 3 and 4 were dosed at 5 μg/kg by the subcutaneous route usingrecombinant and synthetic PTH, respectively. The excipients used for thesubcutaneous route were the same as FORTEO® marketed product for bothgroups.

The mean C_(max) was 1,921.13, 2,559.28, 1,538.10 and 2,526.43 pg/mL forGroup 1, 2, 3 and 4, respectively. The mean T_(max) was 35, 20, 20 and10 minutes for group 1, 2, 3 and 4, respectively. The C_(max) for group2 was 1.3, 1.7 fold greater and approximately the same compared togroups 1, 3, and 4, respectively. However, the dose for group 2 was 10fold higher than group 3 and 4. The relative bioavailability for Group 2corrected for dose was 16.6 and 10.0% compared to groups 3 and 4 forC_(max), respectively. The C_(max) for Group 2 was 1.3 fold higher thanGroup 1. The relative bioavailability comparing the subcutaneous routesC_(max) was 61% for group 3 versus group 4.

The mean AUC_(max) was 111,850.81, 123,498.63, 173,992.88 and 194,895.25min*pg/mL for group 1, 2, 3, and 4, respectively. The mean AUC_(inf) was118,022.48, 130,377.44, 177,755.35 and 206,317.05 for group 1, 2, 3 and4 respectively. The relative bioavailability for Group 2 corrected fordose was 7.1 and 6.3% compared to groups 3 and 4 for AUC_(last),respectively. The relative bioavailability for Group 2 corrected fordose was 7.3 and 6.3% compared to groups 3 and 4 for AUC_(inf),respectively. The AUC_(last) and infinity for Group 2 was 1.1 foldhigher than Group 1. The relative bioavailability comparing thesubcutaneous routes AUC_(last) was 89.3% for group 3 versus group 4. Therelative bioavailability comparing the subcutaneous routes AUC_(inf) was86.2% for group 3 versus group 4.

Based on the human pharmacokinetic data that tested the approvedsubcutaneous FORTEO® at a dose of 20 μg, the human dose systemicexposure multiple for Group 2 are approximately 20, 8.4 and 7.4 fold forC_(max), AUC_(last), and AUC_(inf), respectively. The nasal exposure inthe study is approximately 2 fold based on the mean rabbit weight of 2.5kg and nasal surface area of 30 cm² and 80 cm² for rabbits and humans,respectively.

The t_(1/2) of PTH₁₋₃₄ ranges from approximately 43-55 minutes for allgroups. The mean t_(max) was 35, 20, 20 and 10 minutes for groups 1, 2,3, and 4, respectively. Kel was 0.018, 0.017, 0.016 and 0.014 for groups1, 2, 3, and 4, respectively. No adverse clinical signs were observedfollowing dosing of any formulation.

On a C_(max) and AUC basis the relative bioavailability of formulationof the invention was approximately 10%. Other intranasal studiesconducted in rabbits with PYY₃₋₃₆ as the active with a similarformulation matrix also had an approximate 19% bioavailability and whentested in humans resulted in a similar bioavailability. Therefore, humanintranasal dose is 200 μg in contrast to the FORTEO® subcutaneous doseof 20 μg.

Even though formulation of the invention had a higher C_(max), AUC and afaster T_(max), Group 1 had an unexpected high absorption rate ascompared to Group 2, 3 and 4. consistent with in-vitro studies.

Synthetic PTH₁₋₃₄ has a higher absorption rate than the recombinantPTH₁₋₃₄ given by the subcutaneous route. The levels reached in thisstudy with formulation of the invention are approximately 20 and 8.4fold for C_(max) and AUC_(last) based on a human dose of 20 μg. Based ona nasal surface area basis, the doses tested in this study areapproximately 2 fold. Therefore, the doses chosen in the 14 day toxicitystudies of 50 and 250 μg give an appropriate comparison to the humandose for nasal and systemic exposure.

EXAMPLE 6 PK Study of PTH₁₋₃₄

This study was conducted to determine the pharmacokinetic profile ofteriparatide (human parathyroid hormone 1-34 (hPTH₁₋₃₄)) in selectedformulations following intranasal administration in the rabbit.

The overall study design is provided in Table 5. The dose level wasselected based on previous studies with teriparatide dosed viaintranasal instillation.

Each animal was dosed by intranasal instillation into the left nare.Blood samples were taken from the marginal ear vein, pre-dose and 5, 10,20, 30, 45 minutes and 1 (60 minutes), 2 (120 minutes) and 4 hours (240minutes) post-dose. Blood sampling and handling were conducted perprotocol, with no deviations that were considered to impact samplequality. Blood samples were collected with a protease inhibitor(Aprotinin), processed for harvest of serum and plasma, and frozen andstored at −70° C. until analyzed.

Five nasal formulations of teriparatide were evaluated in the study. Thevehicle composition for each formulation is provided in Table 6. Thenasal formulations of the invention were manufactured at NastechPharmaceutical Company Inc. (Bothell, Wash.).

An enzyme immunoassay was developed (serum and plasma) and validated(serum) to measure the concentration of human PTH₁₋₃₄ in blood samplesfrom rabbit. Study samples, Standards, and Quality Control samples wereassayed using a modified Human Bioactive PTH₁₋₃₄ ELISA kit. Standards,Samples, and Quality Control samples were added to streptavidin coatedstrip wells, with each sample analyzed in duplicate. Samples wereincubated with a mixture of biotinylated anti-human PTH₁₋₃₄ antibody andHRP-conjugated anti-human PTH₁₋₃₄ antibody. The plate was washed withthe kit wash solution and TMB substrate solution was added to each well.Color was allowed to develop for 10 minutes before the reaction wasstopped by the addition of 1 M sulfuric acid to each well. The OD₄₅₀ foreach well was measured on an absorbance plate reader, and theconcentration was calculated by interpolation from the standard curve.Assay performance was monitored with Quality Control samples. The assayLower Limit of Quantification (LLOQ) was determined to be 7.8 pg/mLusing human PTH₁₋₃₄ as the standard analyte and normal rabbit serum orplasma.

Due to species similarity between rabbit and human parathyroid hormones,it was anticipated the assay would detect endogenous (i.e., rabbit)parathyroid hormone and rabbit-PTH₁₋₃₄. Pharmacokinetic calculations asdescribed above.

Pre-dose concentrations of PTH (assumed to represent rabbit parathyroidhormone or its fragments) in serum or plasma were generally below 100pg/mL, with several samples determined to be <62.4 pg/mL. Limitations onsample volume precluded repeated analysis to obtain definitive resultsor the assay LLOQ. The value provided is the lowest value (or estimatedvalue) obtained at which no further analysis was possible.

Individual animal serum and plasma values for teriparatide exceeded 100pg/mL at the 5 through 60 minute time points. At the final time points,particularly 240 minutes, the majority of the animals had teriparatideconcentrations of <31.2 pg/mL (the last point at which sample volume wasinsufficient for further evaluation). Thus the absorption andelimination phases for teriparatide were captured by the sampling timeframe employed for the study.

Formulation 1/Group 1

The C_(max), t_(1/2), and AUC_(last) results for serum and plasma of theGroup 1 animals greatly exceed the expected values based on dose level.Pre-dose values for each of the four animals in Group 1 were within theexpected range, indicating the animals endogenous levels were not afactor in this observation. Animals #931 had teriparatide values inserum and plasma at the 5- and 10-minute that that were within theexpected range. The 5-minute time point for Animal #932 was within theexpected range, while at 10-minutes post-dose the assay value for plasmawas >250,000 pg/mL. Subsequent time points for both animals were foundto have concentrations of >30,000 pg/mL in serum or plasma. For Animals#933 and #934, all post-dose time points for serum and plasma were foundto have teriparatide concentrations that were >30,000 pg/mL. As aresult, C_(max) was >45,000 pg/mL and the t_(1/2) was >300 minutes forthe majority of animals. Mean AUC_(last) was >7,000,000 min*pg/mL.

The mean T_(max) of 29 and 20 minutes for serum and plasma,respectively, were consistent with the expected range for intranasaldosing. However, the measured C_(max) of 30,000 to almost 50,000 pg/mLwere an order of magnitude high than the expected maximal concentrationsof 3000 to 5000 pg/mL.

Formulation 2/Group 2

In serum, the group mean C_(max) and T_(max) were 4396 pg/mL and 36minutes, respectively. The group mean t_(1/2) was 70.7 minutes, however,this was influenced by Animal #935 in which the calculated t_(1/2) was198 minutes; the t_(1/2) for the other three animals was 37.7, 22.7, and24.6 minutes. The mean AUC_(last) was 307213 min*pg/mL. The mean C_(max)and AUC_(last) in plasma were 3819 pg/mL and 105144 min*pg/mL,respectively. T_(max) was estimated to be 9 minutes and the t_(1/2) was66.9 minutes.

Formulation 3/Group 3

C_(max), T_(max), t_(1/2), and AUC_(last) in serum were 1224 pg/mL, 9minutes, 53 minutes, an 35111 min*pg/mL, respectively. In plasma,C_(max), T_(max), ^(and) AUC_(last) were 1102 pg/mL, 8 minutes, and53283 min*pg/mL, respectively. The mean t_(1/2) was estimated to be 99.7mutes, however, this was attributable to a long t_(1/2) (216.6 minutes)for Animal #939.

Formulation 4/Group 4

In serum, C_(max), T_(max), t_(1/2), and AUC_(last) were 653 pg/mL, 35minutes, 22.3 minutes, and 30807 min*pg/mL, respectively. In plasma,these same parameters were 915 pg/mL, 35 minutes, 73.9 minutes, and56383 min*pg/mL, respectively.

Formulation 5/Group 5

In serum, C_(max), T_(max), t_(1/2), and AUClast were 549 pg/mL, 45minutes, 57.6 minutes, and 41489 min*pg/mL, respectively. In plasma,these same parameters in serum, and 734 pg/mL, 41 minutes, 48.8 minutes,and 23710.9 min*pg/mL, respectively.

The post-dose data for Group 1 demonstrate the unexpected highconcentrations and absence of a clear elimination phase. This would beconsistent with a contamination of a common solution in the collectionprocedure, and concentrations that are independent of doseadministration.

For Groups 3 and 5 the mean concentration of teriparatide at each timepoint post-dose was similar between serum and plasma. The shape of theconcentration vs. time curves was also similar between serum and plasma.In Group 2, peak concentrations of teriparatide were similar is serumand plasma, but serum was noted with higher concentrations at the lattertime points. For Group 4, plasma concentrations of teriparatide werehigher at the latter time points, as compared to serum; similarconcentrations of were found in serum and plasma at the early timepoints after dosing. Overall, these results suggest that either plasmaor serum may be an appropriate matrix for the determination ofteriparatide concentrations after dose administration.

The data for Formulation 1/Group 1 was intended to be the set used forcalculation of the comparative bioavailability for the otherformulation, however, with consideration of the data obtained for thisgroup any comparison was considered not to be appropriate. As analternative, data from previous studies, above, were compiled to obtaina value for C_(max) and AUC_(last). Although the formulation componentsfor these studies were slightly different, the primarycomponents—methyl-β cyclodextrin, phosphatidylcholine didecanoyl,edetate disodium, and sodium benzoate—were present at similarconcentrations to Formulation 1.

Using the compiled value for C_(max) and AUC_(last), the comparativebioavailability of teriparatide for each intranasal formulation/groupwas calculated. The C_(max) and AUC_(last) for Formulation 2/Group 2were approximately equal to (serum) or slightly greater (plasma) thanthe compiled values, indicating comparable bioavailability. The primarydifference for Formulation 2 was the concentration ofphosphatidylcholine didecanoyl was decreased to 0.1 mg/mL (from 1.0mg/mL).

Formulation 3 was dosed at half the dose volume, but contained 6.6 mg/mLteriparatide to achieve the same total dose. The concentration ofmethyl-β cyclodextrin, phosphatidylcholine didecanoyl, and edetatedisodium were increased; sodium benzoate remained at the sameconcentration. The comparative bioavailability for Formulation 3 wasapproximately 40% for both serum and plasma.

Formulation 4 contains the components at the concentrations listed forFORTEO®, a subcutaneously delivered form of teriparatide.

Formulation 5 was this formulation with m-cresol removed. Relativebioavailability for Group 4 (Formulation 4) and Group 5 (Formulation 5)was approximately 30% -35% for serum or plasma. The data suggest thatthe presence of m-cresol did not have a significant impact ofteriparatide bioavailability.

These results show that the concentration of phosphatidylcholinedidecanoyl can be decreased from 1.0 mg/mL to 0.1 mg/mL withoutsignificantly decreasing bioavailability. Increasing the concentrationof teriparatide and the absorption enhancers had an apparent negativeimpact upon the bioavailability. The cause for this drop may be relatedto reduction in surface area of contact with the nasal mucosa due toreduced volume. In the absence of absorption enhancers, the relativebioavailability was further decreased. This decreased bioavailabilitywas expected due to the absence of any specific permeation enhancers,and is also consistent with in vitro permeation data. Subcutaneousinjection is the route of administration most often used for nonclinicalstudies with teriparatide, and is the only route approved for clinicaluse. For Group 2, a relative bioavailability in the range of 5 to 17% isconsistent previous estimate of 6-10%. Increasing the concentration ofteriparatide and the absorption enhancers (Group 3) decreased therelative bioavailability to 2-5%. In the absence of absorption enhancers(groups 4 and 5) the relative bioavailability for teriparatide wasapproximately 2-3%.

EXAMPLE 7 PK Study of PTH₁₋₃₄ in Humans

The primary objectives of this study are to: evaluate the absorption ofthree formulations of the invention in a nasal spray at two dose levels;evaluate the safety of these formulations at two dose levels and tocompare the bioavailability of Forsteo given subcutaneously with theseformulations at two dose levels.

This is a phase I, crossover, dose ranging study involving 6 healthymale and 6 healthy female volunteers. There are five study periods asfollows:

Period 1: All 12 subjects receive Forsteo 20 μg subcutaneously.

Period 2: 6 (3 male and 3 female) subjects receive 500 μg of Formulation#2 and 6 (3 male and 3 female) subjects receive 500 μg of Formulation#3. The results of a total calcium level drawn four hours after dosingare used to determine the doses of these Formulations to be administeredduring Periods 4 and 5 as follows:

If none of the six subjects who receive a given Formulation have a totalcalcium level≧3 mmol/L (12.0 mg/dL) at four hours post dose, then alldoses of that Formulation in periods 4 or 5 are at 1000 μg.

If a single subject of the six who receive a given Formulation has atotal calcium level≧3 mmol/L (12.0 mg/dL) at four hours post dose, thatsubject receive only a 500 μg dose of Formulations 2 and 3 duringPeriods 4 and 5.

If 2 or more subjects of the six who receive a given Formulation have atotal calcium level≧3 mmol/L (12.0 mg/dL) at four hours post dose, allsubjects receive a 500 μg dose of that Formulation during Periods 4 or5.

Period 3: All subjects receive 1000 μg of Formulation #1.

Period 4: All subjects receive a dose of Formulation #2 at either 500 μgor 1000 μg as described above.

Period 5: All subjects receive a dose of Formulation #3 at either 500 μgor 1000 μg as described above.

Subjects are confined to the study center from Day-1 until all studyactivities have been completed on Dosing Day #5. Safety assessmentsinclude vital signs, clinical laboratory evaluations, nasal toleranceand adverse events.

Blood samples for PK analysis of teriparatide levels are collected viaan indwelling catheter and/or via direct venipuncture. These sampleswill be collected at 0 (i.e., pre-first-dose), 5, 10, 15, 30, 45, 60, 90minutes and 2, 3, and 4 hours post-dose.

At each time point, 7 mL of blood will be collected. Plasmaconcentrations of teriparatide are determined using a validatedanalytical procedure.

Although the foregoing invention has been described in detail by way ofexample for purposes of clarity of understanding, it will be apparent tothe artisan that certain changes and modifications are comprehended bythe disclosure and may be practiced without undue experimentation withinthe scope of the appended claims, which are presented by way ofillustration not limitation.

TABLE 1 PTH Formulation Composition Formula- PTH₁₋₃₄ tions Per 100 mlSample Mucosal Delivery Enhancing Agent A 60 μg Phosphate-bufferedsaline (0.8%) pH 7.4 (Control 1) B 60 μg Phosphate-buffered saline(0.8%) pH 5.0 (Control 2) C 60 μg L-Arginine (10% w/v) D 60 μgPoly-L-Arginine (0.5% w/v) E 60 μg Gamma-Cyclodextrin (1% w/v) F 60 μgα-Cyclodextrin (5% w/v) G 60 μg Methyl-β-Cyclodextrin (3% w/v) H 60 μgn-Capric Acid Sodium (0.075% w/v) I 60 μg Chitosan (0.5% w/v) J 60 μgL-α-phosphatidilcholine didecanyl (3.5% w/v) K 60 μgS-Nitroso-N-Acetyl-Penicillamine (0.5% w/v) L 60 μgPalmotoyl-DL-Carnitine (0.02% w/v) M 60 μg Pluronic-127 (0.3% w/v) N 60μg Sodium Nitroprusside (0.3% w/v) O 60 μg Sodium Glycocholate (1% w/v)P 60 μg F1: Gelatin, DDPC, MBCD, EDTA F 1 L-α-phosphatidilcholinedidecanyl (0.5% w/v) Methyl β Cyclodextrin (3% w/v) EDTA (0.1% w/v, Inf.Conc. 0.5 M) Gelatin (0.5% w/v)

TABLE 2 Theoretical Weight Ingredient Name g/100 mL (Grams) PTH₁₋₃₄, GMPgrade 0.022 0.0066 Chlorobutanol, NF (anhydrous) 0.50 1.25Methyl-β-Cyclodextrin 4.50 11.25 L-α-Phosphatidylcholine Didecanoyl 0.100.25 Edetate Disodium, USP 0.10 0.25 Sodium Citrate dihydrate, USP0.1800 0.45 Citric acid anhydrous, USP 0.0745 0.1863 Lactosemonohydrate, NF 0.90 2.25 Sorbitol, NF 1.82 4.55 Hydrochloric acid, NF TAP* TAP Sodium Hydroxide, USP TAP TAP Sterile Water for Irrigation,USP 94.03** 235.075

TABLE 3 Formula for PTH1-34 Nasal Spray solution Theoretical WeightIngredient Name g/100 mL (Grams) Chlorobutanol, NF (anhydrous) 0.50 1.25Methyl-β-Cyclodextrin 4.50 11.25 L-α-Phosphatidylcholine Didecanoyl 0.100.25 Edetate Disodium, USP 0.10 0.25 Sodium Citrate dehydrate, USP 0.180.45 Citric acid anhydrous, USP 0.0745 0.1863 Lactose monohydrate, NF0.90 2.25 Sorbitol, NF 1.82 4.55 Hydrochloric acid, NF TAP TAP Sodiumhydroxide, USP TAP TAP Sterile Water for Irrigation, USP 94.03* 235.075TAP = To adjust pH. *Using experimentally determined diluent density of1.022 g/mL

TABLE 4 Dosing Groups for Toxicokinetic Animals Route of Dose Dose DoseAdministration Conc Vol Level Group Animals Formulation (mg/mL) (mL/kg)(μg/kg) 1 4M Intranasal 3.33 0.015 50 (Bachem PTH Marketed Formulation)2 4M Intranasal 3.33 0.015 50 (Bachem PTH Nastech Formulation) 3 4MSubcutaneous (Sub-Q) 0.025 0.2 5 (Marketed Product) 4 4M Subcutaneous(Sub-Q) 0.025 0.2 5 (Bachem PTH Marketed Formulation)

TABLE 5 Dosing Groups for Pharmacokinetic Evaluation Route ofTeriparatide Dose Teriparatide Study Ani- Administration ConcentrationVolume Dose Level Groups mals (Formulation) (mg/mL) (mL/kg) (μg/kg) 1 4MIntranasal 3.33 0.015 50 (Formulation 1) 2 4M Intranasal 3.33 0.015 50(Formulation 2) 3 4M Intranasal 6.6 0.0075 50 (Formulation 3) 4 4MIntranasal 3.33 0.015 50 (Formulation 4) 5 4M Intranasal 3.33 0.015 50(Formulation 5)

TABLE 6 Vehicle Composition for Formulations 1-5 Component mg/mL (% w/w)Formulation 1 Methyl-β Cyclodextrin 45 (4.5) Phosphatidylcholinedidecanoyl (DDPC) 1.0 (0.1) Edetate Disodium, USP 1.0 (0.1) SodiumBenzoate, NF 4.0 (0.4) Sodium Hydroxide, USP TAP Hydrochloric Acid, NFTAP Sterile Water for Irrigation, USP q.s pH = 4.5 Formulation 2Methyl-β Cyclodextrin 45 (4.5) Phosphatidylcholine didecanoyl (DDPC) 0.1(0.01) Edetate Disodium, USP 1.0 (0.1) Sodium Benzoate, NF 4.0 (0.4)Sodium Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Water forIrrigation, USP q.s pH = 4.5 Formulation 3 Methyl-β Cyclodextrin 90(9.0) Phosphatidylcholine didecanoyl (DDPC) 2.0 (0.2) Edetate Disodium,USP 2.0 (0.2 Sodium Benzoate, NF 4.0 (0.4) Sodium Hydroxide, USP TAPHydrochloric Acid, NF TAP Sterile Water for Irrigation, USP q.s pH = 4.5Formulation 4 Mannitol, USP 30.8 (3.08) M-Cresol 3.0 (0.3) GlacialAcetic Acid, USP 0.42 (0.042) Sodium Acetate, Anhydrous, USP 0.1 (0.01)Sodium Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Water forIrrigation, USP q.s pH = 4.0 Formulation 5 Mannitol, USP 30.8 (3.08)Glacial Acetic Acid, USP 0.42 (0.042) Sodium Acetate, Anhydrous, USP 0.1(0.01) Sodium Hydroxide, USP TAP Hydrochloric Acid, NF TAP Sterile Waterfor Irrigation, USP q.s pH = 4.0 TAP = added to adjust final pH

1. A pharmaceutical composition for intranasal delivery comprising anaqueous mixture of a PTH, a cyclodextrin, a phospholipid, ethylenediamine tetraacetic acid, a polyol, and a preservative, wherein thecomposition has at least 6% bioavailability of the PTH upon intranasaladministration of the composition to a human.
 2. The pharmaceuticalcomposition of claim 1, wherein the composition has at least 8%bioavailability of the PTH upon intranasal administration of thecomposition to a human.
 3. The pharmaceutical composition of claim 1,wherein the PTH is selected from SEQ ID NO:1, SEQ ID NO:2, and SEQ IDNO:3.
 4. The pharmaceutical composition of claim 1, wherein thecyclodextrin is hydroxypropyl-β-cyclodextrin,sulfobutylether-β-cyclodextrin, or methyl-β-cyclodextrin.
 5. Thepharmaceutical composition of claim 1, wherein the cyclodextrin ismethyl-β-cyclodextrin at a concentration of 4.5% (w/w).
 6. Thepharmaceutical composition of claim 1, wherein the phospholipid isdidecanoylphosphatidylcholine (DDPC).
 7. The pharmaceutical compositionof claim 1, wherein the polyol is selected from sucrose, mannitol,sorbitol, lactose, L-arabinose, D-erythrose, D-ribose, D-xylose,D-mannose, trehalose, D-galactose, lactulose, cellobiose, andgentibiose.
 8. The pharmaceutical composition of claim 1, wherein thepreservative is chlorobutanol or sodium benzoate.
 9. The pharmaceuticalcomposition of claim 1, wherein the composition has a pH of from about 3to about
 6. 10. The pharmaceutical composition of claim 1, wherein thecomposition is in the form of liquid droplets.
 11. The pharmaceuticalcomposition of claim 10, wherein the liquid droplets have an averagevolume-mean particle size (Dv,50) of from about 1 micron to about 1000microns.
 12. The pharmaceutical composition of claim 10, where in theliquid droplets have an average volume-mean particle size (Dv,50) offrom about 5 microns to about 500 microns.
 13. The pharmaceuticalcomposition of claim 10, where in the liquid droplets have an averagevolume-mean particle size (Dv,50) of from about 10 microns to about 100microns.
 14. A method for treating osteoporosis or osteopenia in a humansubject comprising administering intranasally to the human apharmaceutical composition of claim
 1. 15. The method of claim 14,wherein upon intranasal administration to the human subject the PTH hasa maximum serum concentration, Cmax, of about 100 pg/mL or greater. 16.The method of claim 14, wherein the dose of PTH administered to thehuman subject is from about 1 microgram to about 2000 microgram.
 17. Amethod for preventing the onset of osteoporosis or osteopenia in a humansubject comprising administering intranasally to the human apharmaceutical composition of claim 1.